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The integrin LFA-1 is crucial for T-cell/ APC interactions and sensitive recognition of antigens. Precise nanoscale organization and valency regulation of LFA-1 are mandatory for an appropriate function of the immune system. While the inside-out signals regulating the LFA-1 affinity are well described, the molecular mechanisms controlling LFA-1 avidity are still not fully understood. Here, we show that activation of the actin-bundling protein L-plastin (LPL) through phosphorylation at serine-5 enables the formation of clusters containing LFA-1 in high-affinity conformation. Phosphorylation of LPL is induced by an nPKC-MEK-p90RSK pathway and counter-regulated by the serine-threonine phosphatase PP2A. Interestingly, recruitment of LFA-1 into the T-cell/APC contact zone is not affected by LPL phosphorylation. Instead, for this process, activation of the actin-remodeling protein cofilin through dephosphorylation is essential. Together, this study reveals a dichotomic spatial regulation of LFA-1 clustering and microscale movement in T-cells by two different actin-binding proteins, LPL and cofilin.

ObjectivesNeutrophils are typically the most abundant leukocyte in arthritic synovial fluid. We sought to understand changes that occur in neutrophils as they migrate from blood to joint.MethodsWe performed RNA sequencing of neutrophils from healthy human blood, arthritic blood, and arthritic synovial fluid, comparing transcriptional signatures with those from murine K/BxN serum transfer arthritis. We employed mass cytometry to quantify protein expression and sought to reproduce the synovial fluid phenotypeex vivoin cultured healthy blood neutrophils.ResultsBlood neutrophils from healthy donors and patients with active arthritis exhibited largely similar transcriptional signatures. By contrast, synovial fluid neutrophils exhibited more than 1,600 differentially expressed genes. Gene signatures identified a prominent response to interferon gamma (IFNγ), as well as to tumor necrosis factor, interleukin 6, and hypoxia, in both humans and mice. Mass cytometry also found healthy and arthritic donor blood neutrophils largely indistinguishable but revealed a range of neutrophil phenotypes in synovial fluid defined by downregulation of CXCR1 and upregulation of FcγRI, HLA-DR, PD-L1, ICAM-1 and CXCR4. Reproduction of key elements of this signature in cultured blood neutrophils required both IFNγ and prolonged culture.ConclusionsCirculating neutrophils from arthritis patients resemble those from healthy controls, but joint fluid cells exhibit a network of changes, conserved across species, that implicate IFNγ response and aging as complementary drivers of the synovial neutrophil phenotype.KEY MESSAGESWhat is already known about this subject?Neutrophils are central in the effector phase of inflammatory arthritis but their phenotypic heterogeneity in inflamed synovial fluid is poorly understood.What does this study add?RNA-seq and mass cytometry identify a hallmark phenotype of neutrophils in synovial fluid consisting of upregulated ICAM-1, HLA-DR, PD-L1, Fc receptors and CXCR4.Transcriptomics highlight an IFNγ response signature conserved across humans and mice.In vitro experiments implicate aging and IFNγ as complementary factors orchestrating the synovial fluid neutrophil phenotype.How might this impact on clinical practice or future developments?Understanding the specific features of neutrophils in the arthritic joint may disclose opportunities for safe therapeutic targeting of this lineage.

Cytokines released during chronic inflammatory diseases induce pro-inflammatory properties in polymorphonuclear neutrophils (PMN). Here we show that in vitro cytokine treatment leads to the development of a subgroup of human PMN expressing CCR5, termed CCR5+ cytokine-induced PMN (CCR5+ cPMN). Auto/paracrine TNF signaling increases intracellular neutrophil elastase (ELANE) abundance and induces NETosis in CCR5+ cPMN. Triggering of CCR5 amplifies NETosis. Membranous TNF (mTNF) outside-in signaling induces the formation of reactive oxygen species, a known activator of NETosis. In vivo, we find an increased number of CCR5+ cPMN in the peripheral blood and inflamed lamina propria of patients with ulcerative colitis (UC) but not Crohn’s disease (CD). Notably, failure of anti-TNF therapy is associated with higher frequencies of CCR5+ cPMN. In conclusion, we identify a phenotype of pro-NETotic, CCR5 positive PMN present in inflamed tissue in vivo and inducible in vitro. These cells may reflect an important component of tissue damage during chronic inflammation and could be of diagnostic value.

The understanding of the tumor microenvironment (TME) has been expanding in recent years in the context of interactions among different cell types, through direct cell–cell communication as well as through soluble factors. It has become evident that the development of a successful antitumor response depends on several TME factors. In this context, the number, type, and subsets of immune cells, as well as the functionality, memory, and exhaustion state of leukocytes are key factors of the TME. Both the presence and functionality of immune cells, in particular T cells, are regulated by cellular and soluble factors of the TME. In this regard, one fundamental reason for failure of antitumor responses is hijacked immune cells, which contribute to the immunosuppressive TME in multiple ways. Specifically, reactive oxygen species (ROS), metabolites, and anti-inflammatory cytokines have central roles in generating an immunosuppressive TME. In this review, we focused on recent developments in the immune cell constituents of the TME, and the micromilieu control of antitumor responses. Furthermore, we highlighted the current challenges of T cell-based immunotherapies and potential future strategies to consider for strengthening their effectiveness.

The interplay between keratinocytes and immune cells, especially T cells, plays an important role in the pathogenesis of chronic inflammatory skin diseases. During psoriasis, keratinocytes attract T cells by releasing chemokines, while skin-infiltrating self-reactive T cells secrete proinflammatory cytokines, e.g., IFNγ and IL-17A, that cause epidermal hyperplasia. Similarly, in chronic graft-versus-host disease, allogenic IFNγ-producing Th1/Tc1 and IL-17-producing Th17/Tc17 cells are recruited by keratinocyte-derived chemokines and accumulate in the skin. However, whether keratinocytes act as nonprofessional antigen-presenting cells to directly activate naive human T cells in the epidermis remains unknown. Here, we demonstrate that under proinflammatory conditions, primary human keratinocytes indeed activate naive human T cells. This activation required cell contact and costimulatory signaling via CD58/CD2 and CD54/LFA-1. Naive T cells costimulated by keratinocytes selectively differentiated into Th1 and Th17 cells. In particular, keratinocyte-initiated Th1 differentiation was dependent on costimulation through CD58/CD2. The latter molecule initiated STAT1 signaling and IFNγ production in T cells. Costimulation of T cells by keratinocytes resulting in Th1 and Th17 differentiation represents a new explanation for the local enrichment of Th1 and Th17 cells in the skin of patients with a chronic inflammatory skin disease. Consequently, local interference with T cell–keratinocyte interactions may represent a novel strategy for the treatment of Th1 and Th17 cell-driven skin diseases.

NF-κB inducing kinase (NIK) is the key protein of the non-canonical NF-κB pathway and is important for the development of lymph nodes and other secondary immune organs. We elucidated the specific role of NIK in T cells using T-cell specific NIK-deficient (NIKΔT) mice. Despite showing normal development of lymphoid organs, NIKΔT mice were resistant to induction of CNS autoimmunity. T cells from NIKΔT mice were deficient in late priming, failed to up-regulate T-bet and to transmigrate into the CNS. Proteomic analysis of activated NIK-/- T cells showed de-regulated expression of proteins involved in the formation of the immunological synapse: in particular, proteins involved in cytoskeleton dynamics. In line with this we found that NIK-deficient T cells were hampered in phosphorylation of Zap70, LAT, AKT, ERK1/2 and PLCγ upon TCR engagement. Hence, our data disclose a hitherto unknown function of NIK in T-cell priming and differentiation.

Neutrophils or polymorphonuclear cells (PMN) eliminate bacteria via phagocytosis and/or NETosis. Apart from these conventional roles, PMN also have immune-regulatory functions. They can transdifferentiate and upregulate MHCII as well as ligands for costimulatory receptors which enables them to behave as antigen presenting cells (APC). The initial step for activating T-cells is the formation of an immune synapse between T-cells and antigen-presenting cells. However, the immune synapse that develops at the PMN/T-cell contact zone is as yet hardly investigated due to the non-availability of methods for analysis of large number of PMN interactions. In order to overcome these obstacles, we introduce here a workflow to analyse the immune synapse of primary human PMN and T-cells using multispectral imaging flow cytometry (InFlow microscopy) and super-resolution microscopy. For that purpose, we used CD3 and CD66b as the lineage markers for T-cells and PMN, respectively. Thereafter, we applied and critically discussed various "masks" for identification of T-cell PMN interactions. Using this approach, we found that a small fraction of transdifferentiated PMN (CD66b+CD86high) formed stable PMN/T-cell conjugates. Interestingly, while both CD3 and CD66b accumulation in the immune synapse was dependent on the maturation state of the PMN, only CD3 accumulation was greatly enhanced by the presence of superantigen. The actin cytoskeleton was weakly rearranged at the PMN side on the immune synapse upon contact with a T-cell in the presence of superantigen. A more detailed analysis using super-resolution microscopy (structured-illumination microscopy, SIM) confirmed this finding. Together, we present an InFlow microscopy based approach for the large scale analysis of PMN/T-cell interactions and - combined with SIM - a possibility for an in-depth analysis of protein translocation at the site of interactions.

T-cells need to be tightly regulated during their activation and effector phase to assure an appropriate defence against cancer or pathogens and - vice versa - to avoid autoimmune reactions. Regulatory signals are provided via the immune synapse between T-cells and antigen-presenting cells (APCs) or target cells. The stability and kinetics of immune synapse formation is critical for proper T-cell functions. It requires dynamic rearrangements of the actin cytoskeleton necessary for organized spatio-temporal redistribution of receptors and adhesion molecules. We identified glucocorticoid-sensitive phosphorylation of serine 5 on the actin-bundling protein L-plastin as one important signalling event for this regulation. Using imaging flow cytometry as well as confocal and super-resolution microscopy we showed that L-plastin relocalizes to the immune synapse upon antigen encounter, where it associates with the β2-subunit of LFA-1 (CD11a/CD18). Interfering with L-plastin expression or activation leads to a defective LFA-1 recruitment and unstable T-cell/APC contacts. Consequently, the lack of L-plastin diminishes T-cell activation, proliferation and proximal effector responses such as cytokine production. On the other hand, a pro-oxidative milieu leads to prolonged activation of L-plastin resulting in a stronger enrichment of LFA-1 in the cytolytic immune synapse. Concomitant stabilization of conjugates formed by cytotoxic T-cells (CTLs) and their target cells impairs the ability of CTLs to kill more than one target cells (serial killing), which de facto leads to a downregulation of T-cell cytotoxicity. Together, we demonstrate that activation and spacial distribution of L-plastin regulates the maturation and stability of activating and cytolytic immune synapses important for T-cell activation and effector functions.

Background The enteric nervous system (ENS), a complex network of neurons and glial cells, coordinates major gastrointestinal functions. Impaired development or secondary aberrations cause severe enteric neuropathies. Neural crest-derived stem cells as well as enteric neuronal progenitor cells, which form enteric neurospheres, represent a promising tool to unravel molecular pathomechanisms and to develop novel therapy options. However, so far little is known about the detailed cellular composition and the proportional distribution of enteric neurospheres. Comprehensive knowledge will not only be essential for basic research but also for prospective cell replacement therapies to restore or to improve enteric neuronal dysfunction. Methods Human enteric neurospheres were generated from three individuals with varying age. For detailed molecular characterization, nCounter target gene expression analyses focusing on stem, progenitor, neuronal, glial, muscular, and epithelial cell markers were performed. Corresponding archived paraffin-embedded individuals' specimens were analyzed accordingly. Key results Our data revealed a remarkable molecular complexity of enteric neurospheres and archived specimens. Amongst the expression of multipotent stem cell, progenitor cell, neuronal, glial, muscle and epithelial cell markers, moderate levels for the pluripotency marker POU5F1 were observed. Furthermore, besides the interindividual variability, we identified highly distinct intraindividual expression profiles. Conclusions & inferences Our results emphasize the assessment of molecular signatures to be essential for standardized use, optimization of experimental approaches, and elimination of potential risk factors, as the formation of tumors. Our study pipeline may serve as a blueprint implemented into the characterization procedure of enteric neurospheres for various future applications.

Patients with the neurological disorder HSAN-I suffer frequent infections, attributed to a lack of pain sensation and failure to seek care for minor injuries. Whether protective CD8(+) T cells are affected in HSAN-I patients remains unknown. Here, we report that HSAN-I-associated mutations in serine palmitoyltransferase subunit SPTLC2 dampened human T cell responses. Antigen stimulation and inflammation induced SPTLC2 expression, and murine T-cell-specific ablation of Sptlc2 impaired antiviralT-cell expansion and effector function. Sptlc2 deficiency reduced sphingolipid biosynthetic flux and led to prolonged activation of the mechanistic target of rapamycin complex 1 (mTORC1), endoplasmic reticulum (ER) stress, and CD8(+) T cell death. Protective CD8(+) T cell responses in HSAN-I patient PBMCs and Sptlc2-deficient mice were restored by supplementing with sphingolipids and pharmacologically inhibiting ER stress-induced cell death. Therefore, SPTLC2 underpins protective immunity by translating extracellular stimuli into intracellular anabolic signals and antagonizes ER stress to promote T cell metabolic fitness.

While several protein serine/threonine kinases control cytokine production by T cells, the roles of serine/threonine phosphatases are largely unexplored. Here, we analyzed the involvement of protein phosphatase 1α (PP1α) in cytokine synthesis following costimulation of primary human T cells. Small interfering RNA (siRNA)-mediated knockdown of PP1α (PP1(KD)) or expression of a dominant negative PP1α (D95N-PP1) drastically diminished interleukin-10 (IL-10) production. Focusing on a key transcriptional activator of human IL-10, we demonstrate that nuclear translocation of NF-κB was significantly inhibited in PP1(KD) or D95N-PP1 cells. Interestingly, knockdown of cofilin, a known substrate of PP1 containing a nuclear localization signal, also prevented nuclear accumulation of NF-κB. Expression of a constitutively active nonphosphorylatable S3A-cofilin in D95N-PP1 cells restored nuclear translocation of NF-κB and IL-10 expression. Subpopulation analysis revealed that defective nuclear translocation of NF-κB was most prominent in CD4(+) CD45RA(−) CXCR3(−) T cells that included IL-10-producing T(H)2 cells. Together these findings reveal novel functions for PP1α and its substrate cofilin in T cells namely the regulation of the nuclear translocation of NF-κB and promotion of IL-10 production. These data suggest that stimulation of PP1α could limit the overwhelming immune responses seen in chronic inflammatory diseases.

Plasma fibronectin is a circulating protein that facilitates phagocytosis by connecting bacteria to immune cells. A fibronectin isoform, which includes a sequence of 90 AA called extra-domain B (EDB), is synthesized de novo at the messenger RNA (mRNA) level in immune cells, but the reason for its expression remains elusive. We detected an 80-fold increase in EDB-containing fibronectin in the cerebrospinal fluid of patients with bacterial meningitis that was most pronounced in staphylococcal infections. A role for this isoform in phagocytosis was further suggested by enhanced EDB fibronectin release after internalization of Staphylococcus aureus in vitro. Using transgenic mouse models, we established that immune cell production of fibronectin contributes to phagocytosis, more so than circulating plasma fibronectin, and that accentuated release of EDB-containing fibronectin by immune cells improved phagocytosis. In line with this, administration of EDB fibronectin enhanced in vitro phagocytosis to a larger extent than plasma fibronectin. This enhancement was mediated by αvβ3 integrin as shown using inhibitors or cells from β3 integrin knockout mice. Thus, we identified both a novel function for EDB fibronectin in augmenting phagocytosis over circulating plasma fibronectin, as well as the mediating receptor. Our data also establish for the first time, a direct role for β3 integrin in bacterial phagocytosis in mammals. Key messages • Fibronectin containing an extra domain called EDB is released in bacterial meningitis. • EDB-containing fibronectin enhances phagocytosis more than plasma fibronectin. • The enhancement is mediated by activation of αvβ3 integrin in the presence of EDB. Electronic supplementary material The online version of this article (doi:10.1007/s00109-015-1373-0) contains supplementary material, which is available to authorized users.

The actin cytoskeleton is a main component to preserve the cell shape. It represents a cellular machinery that enables morphological changes and orchestrates important dynamic cellular functions. Thereby, it supports T-cell migration, immune synapse formation, activation and execution of effector functions. The analysis of actin rearrangements in T-cells is therefore an important field of basic and clinical research. Actin reorganization is traditionally performed using flow cytometry or confocal microscopy. However, while flow cytometry lacks spatial and structural information, confocal microscopy is time consuming and not feasible for the characterization of rare events or of un-purified primary cell populations. Here we describe a methodology to analyze actin rearrangements using InFlow microscopy, which is a hybrid technique consisting of flow cytometric and microscopic features. We show that InFlow microscopy is a valuable tool for quantification of the amount and distribution of F-actin in human T-cells after stimulation with chemokines or antigen-presenting cells.

Innate immune cells can sense hepatitis C virus (HCV)-infected cells and respond with anti-viral actions including secretion of interferons (IFNs). In previous studies, the response of individual innate immune cells against HCV was analyzed in detail. We hypothesized that interaction of multiple innate immune cells increases the magnitude of the immune response and eventually leads to clearance of HCV-infected cells. To investigate this, we co-cultured Huh-7 HCV subgenomic replicon (SGR) cells with peripheral blood mononuclear cells (PBMCs). We confirm secretion of IFNα by plasmacytoid dendritic cells (pDCs) and IFNγ by natural killer (NK) cells in the co-culture setup. Moreover, we observed that also monocytes contribute to the anti-viral response. Flow cytometry and ImageStream analysis demonstrated that monocytes take up material from HCV SGR cells in co-culture with PBMCs. Preceding the uptake, PBMCs caused apoptosis of HCV SGR cells by tumor necrosis factor-related apoptosis inducing ligand (TRAIL) expression on NK cells. We observed that only the interplay of monocytes, pDCs, and NK cells resulted in efficient clearance of HCV SGR cells, while these cell populations alone did not kill HCV SGR cells. Despite similar TRAIL receptor expression on Huh-7 control cells and HCV SGR cells, HCV activated PBMCs specifically killed HCV SGR cells and did not target Huh-7 control cells. Finally, we showed that HCV replicating cells per se are sensitive toward TRAIL-induced apoptosis. Our results highlight the importance of the interplay of different innate immune cells to initiate an efficient, rapid, and specific response against HCV-infected cells.

Cofilin is an actin-binding protein that depolymerizes and/or severs actin filaments. This dual function of cofilin makes it one of the major regulators of actin dynamics important for T-cell activation and migration. The activity of cofilin is spatio-temporally regulated. Its main control mechanisms comprise a molecular toolbox of phospho-, phospholipid, and redox regulation. Phosphorylated cofilin is inactive and represents the dominant cofilin fraction in the cytoplasm of resting human T cells. A fraction of dephosphorylated cofilin is kept inactive at the plasma membrane by binding to phosphatidylinositol 4,5-bisphosphate. Costimulation via the T-cell receptor/CD3 complex (signal 1) together with accessory receptors (signal 2) or triggering through the chemokine SDF1α (stromal cell-derived factor 1α) induce Ras-dependent dephosphorylation of cofilin, which is important for immune synapse formation, T-cell activation, and T-cell migration. Recently, it became evident that cofilin is also highly sensitive for microenvironmental changes, particularly for alterations in the redox milieu. Cofilin is inactivated by oxidation, provoking T-cell hyporesponsiveness or necrotic-like programmed cell death. In contrast, in a reducing environment, even phosphatidylinositol 4,5-bisphosphate -bound cofilin becomes active, leading to actin dynamics in the vicinity of the plasma membrane. In addition to the well-established three signals for T-cell activation, this microenvironmental control of cofilin delivers a modulating signal for T-cell-dependent immune reactions. This fourth modulating signal highly impacts both initial T-cell activation and the effector phase of T-cell-mediated immune responses.

Unlike activated CD4(+) T cells, resting CD4(+) T cells are highly resistant to productive HIV-1 infection. Early after HIV-1 entry, a major block limits reverse transcription of incoming viral genomes. Here we show that the deoxynucleoside triphosphate triphosphohydrolase SAMHD1 prevents reverse transcription of HIV-1 RNA in resting CD4(+) T cells. SAMHD1 is abundantly expressed in resting CD4(+) T cells circulating in peripheral blood and residing in lymphoid organs. The early restriction to infection in unstimulated CD4(+) T cells is overcome by HIV-1 or HIV-2 virions into which viral Vpx is artificially or naturally packaged, respectively, or by addition of exogenous deoxynucleosides. Vpx-mediated proteasomal degradation of SAMHD1 and elevation of intracellular deoxynucleotide pools precede successful infection by Vpx-carrying HIV. Resting CD4(+) T cells from healthy donors following SAMHD1 silencing or from a patient with Aicardi-Goutières syndrome homozygous for a nonsense mutation in SAMHD1 were permissive for HIV-1 infection. Thus, SAMHD1 imposes an effective restriction to HIV-1 infection in the large pool of noncycling CD4(+) T cells in vivo. Bypassing SAMHD1 was insufficient for the release of viral progeny, implicating other barriers at later stages of HIV replication. Together, these findings may unveil new ways to interfere with the immune evasion and T cell immunopathology of pandemic HIV-1.

SummarySignal transduction to nuclear factor-kappa B (NF-κB) involves multiple kinases and phosphorylated target proteins, but little is known about signal termination by dephosphorylation. By RNAi screening, we have identified protein phosphatase 4 regulatory subunit 1 (PP4R1) as a negative regulator of NF-κB activity in T lymphocytes. PP4R1 formed part of a distinct PP4 holoenzyme and bridged the inhibitor of NF-κB kinase (IKK) complex and the phosphatase PP4c, thereby directing PP4c activity to dephosphorylate and inactivate the IKK complex. PP4R1 expression was triggered upon activation and proliferation of primary human T lymphocytes and deficiency for PP4R1 caused sustained and increased IKK activity, T cell hyperactivation, and aberrant NF-κB signaling in NF-κB-addicted T cell lymphomas. Collectively, our results unravel PP4R1 as a previously unknown activation-associated negative regulator of IKK activity in lymphocytes whose downregulation promotes oncogenic NF-κB signaling in a subgroup of T cell lymphomas.

Cytotoxic T-cells (CTLs) play an important role in many immune-mediated inflammatory diseases. Targeting cytotoxicity of CTLs would allow to interfere with immune-mediated tissue destruction. Here we demonstrate that WF-10, a pro-oxidative compound, inhibits CTL-mediated cytotoxicity. WF-10 did not influence early steps of target-cell killing, but impaired the ability of CTLs to detach from the initial target cell and to move to a second target cell. This reduced serial killing was accompanied by stronger enrichment of the adhesion molecule LFA-1 in the cytolytic immune synapse. LFA-1 clustering requires activation of the actin-bundling protein L-plastin and was accordingly diminished in L-plastin knockdown cells. Interestingly, WF-10 likely acts through regulating L-plastin: (I) It induced L-plastin activation through phosphorylation leading to enhanced LFA-1-mediated cell adhesion, and, importantly, (II) WF-10 lost its influence on target-cell killing in L-plastin knockdown cells. Finally, we demonstrate that WF-10 can improve immunosuppression by conventional drugs. Thus, while cyclosporine A alone had no significant effect on cytotoxicity of CTLs, a combination of cyclosporine A and WF-10 blocked target-cell killing synergistically. Together, our findings suggest that WF-10 – either alone or in combination with conventional immunosuppressive drugs – may be efficient to control progression of diseases, in which CTLs are crucially involved.

Fibronectin is a multidomain protein secreted by various cell types. It forms a network of fibers within the extracellular matrix and impacts intracellular processes by binding to various molecules, primarily integrin receptors on the cells. Both the presence of several isoforms and the ability of the various domains and isoforms to bind to a variety of integrins result in a wide range of effects. In vivo findings suggest that fibronectin isoforms produced by the osteoblasts enhance their differentiation. Here we report that the isoform characterized by the presence of extradomain A activates α4β1 integrin and augments osteoblast differentiation. In addition, the isoform containing extradomain B enhances the binding of fibronectin through the RGD sequence to β3-containing integrin, resulting in increased mineralization by and differentiation of osteoblasts. Our study thus reveals novel functions for two fibronectin isoforms and the mediating receptors in osteoblast differentiation.

The amino acid arginine is fundamentally involved in the regulation of the immune response during infection, inflammatory diseases and tumor growth. Arginine deficiency (e.g. due to the myeloid cell enzyme arginase) inhibits proliferation and effector functions of activated T lymphocytes. Here, we studied intracellular mechanisms mediating this suppression of human T lymphocytes. Our proteomic analysis revealed an impaired dephosphorylation of the actin-binding protein cofilin upon T-cell activation in the absence of arginine. We show that this correlates with alteration of actin polymerization and impaired accumulation of CD2 and CD3 in the evolving immunological synapse in T cell-antigen presenting cells conjugates. In contrast, T-cell cytokine synthesis is differentially regulated in human T lymphocytes in the absence of arginine. While the production of certain cytokines (e.g. IFN-γ) is severely reduced, T lymphocytes produce other cytokines (e.g. IL-2) independent of extracellular arginine. MEK and PI3K activity are reciprocally regulated in association with impaired cofilin dephosphorylation. Finally, we show that impaired cofilin dephosphorylation is also detectable in human T cells activated in a granulocyte-dominated purulent micromilieu due to arginase-mediated arginine depletion. Our novel results identify cofilin as a potential regulator of human T-cell activation under conditions of inflammatory arginine deficiency.

Activation of naïve T cells requires costimulation via TCR/CD3 plus accessory receptors, which enables the dynamic rearrangement of the actin cytoskeleton and immune synapse maturation. Signaling events induced following costimulation may thus be valuable targets for therapeutic immunosuppression. Phosphorylation of the actin-bundling protein L-plastin represents such a costimulatory signal in primary human T cells. Phosphorylated L-plastin has a higher affinity toward F-actin. However, the importance of the L-plastin phosphorylation for actin cytoskeleton regulation upon antigen recognition remained unclear. Here, we demonstrate that phosphorylation of L-plastin is important for immune synapse maturation. Thus, expression of nonphosphorylatable L-plastin in untransformed human peripheral blood T cells leads to reduced accumulation of LFA-1 in the immune synapse and to a diminished F-actin increase upon T-cell activation. Interestingly, L-plastin phosphorylation is inhibited by the glucocorticoid dexamethasone. In line with this finding, dexamethasone treatment leads to a reduced F-actin content in stimulated T cells and prevents maturation of the immune synapse. This inhibitory effect of dexamethasone could be reverted by expression of a phospho-mimicking L-plastin mutant. In conclusion, our data introduce costimulation-induced L-plastin phosphorylation as an important event for immune synapse formation and its inhibition by dexamethasone as a novel mode of function of this immunosuppressive glucocorticoid.

Dendritic cells (DCs) are key components of the adaptive immune system contributing to initiation and regulation of T cell responses. T cells continuously scan DCs in lymphoid organs for the presence of foreign antigen. However, little is known about the functional consequences of these frequent T cell–DC interactions without cognate antigen. Here we demonstrate that these contacts in the absence of foreign antigen serve an important function, namely, induction of a basal activation level in T cells required for responsiveness to subsequent encounters with foreign antigens. This basal activation is provided by self-recognition of MHC molecules on DCs. Following DC depletion in mice, T cells became impaired in TCR signaling and immune synapse formation, and consequently were hyporesponsive to antigen. This process was reversible, as T cells quickly recovered when the number of DCs returned to a normal level. The extent of T cell reactivity correlated with the degree of DC depletion in lymphoid organs, suggesting that a full DC compartment guarantees optimal T cell responsiveness. These findings indicate that DCs are specialized cells that not only present foreign antigen, but also promote a “tonic” state in T cells for antigen responsiveness.

Bacteria organised in biofilms are a common cause of relapsing or persistent infections, particularly in patients receiving medical implants such as ventilation tubes, indwelling catheters, artificial heart valves, endoprostheses, or osteosynthesis materials. Bacteria in biofilms are relatively resistant towards antibiotics and biocides, and--according to the current dogma--towards the host defence mechanisms as well. In that context, we addressed the question, how polymorphonuclear neutrophils (PMN), the "first line defence" against bacterial infection, would interact with Staphylococcus aureus biofilms generated in vitro. By time-lapse video microscopy and confocal laser scan microscopy we observed a migration of PMN towards and into the biofilms, as well as clearance of biofilms by phagocytosis. By labelling the bacteria within the biofilm with (3)H thymidine, and by cytofluorometry we could confirm and quantify clearance and phagocytosis of biofilm as well. Of note, the extent of biofilm clearance depended on its maturation state: developing "young" biofilms were more sensitive towards the attack by PMN compared to mature biofilms. In conclusion, contrary to the current dogma, S. aureus biofilms are not inherently protected against the host defence.

SummaryOxidative stress leads to impaired T cell activation. A central integrator of T cell activation is the actin-remodelling protein cofilin. Cofilin is activated through dephosphorylation at Ser3. Activated cofilin enables actin dynamics through severing and depolymerization of F-actin. Binding of cofilin to actin is required for formation of the immune synapse and T cell activation. Here, we showed that oxidatively stressed human T cells were impaired in chemotaxis- and costimulation-induced F-actin modulation. Although cofilin was dephosphorylated, steady-state F-actin levels increased under oxidative stress conditions. Mass spectrometry revealed that cofilin itself was a target for oxidation. Cofilin oxidation induced formation of an intramolecular disulfide bridge and loss of its Ser3 phosphorylation. Importantly, dephosphorylated oxidized cofilin, although still able to bind to F-actin, did not mediate F-actin depolymerization. Impairing actin dynamics through oxidation of cofilin provides a molecular explanation for the T cell hyporesponsiveness caused by oxidative stress.

Rearrangements in the actin cytoskeleton play a pivotal role for costimulation-induced formation of the immunological synapse and T cell activation. Yet, little is known about the actin-binding proteins that link costimulation to rearrangements in the actin cytoskeleton. Here we demonstrate that phosphorylation of the actin bundling protein L-plastin in response to costimulation through TCR/CD3 plus CD2 or CD28, respectively, is important for the activation of human peripheral blood T lymphocytes (PBT). Mass spectrometry and site-directed mutagenesis revealed that Ser5 represents the only phospho-acceptor site of L-plastin in PBT. Wild-type L-plastin (wt-LPL) and a non-phosphorylatable 5A-L-plastin (5A-LPL) equally relocalized to the immunological synapse between PBT and APC. Yet importantly, cells expressing 5A-LPL showed a significantly lower expression of the T cell activation molecules CD25 and CD69 on the cell surface than cells expressing wt-LPL. This effect is due to a failure in the transport of CD25 and CD69 to the cell surface since the total amount of these proteins within the cells remained unchanged. In conclusion, phosphorylation of the actin bundling protein L-plastin represents a so-far-unknown mechanism by which costimulation controls the transport of activation receptors to the T cell surface.

The leukocyte specific actin-binding protein L-plastin is aberrantly expressed in several nonhematopoetic malignant tumors. However, little is known about the functional consequences of L-plastin expression. Here, we investigated the function of L-plastin in human malignant melanoma cells. Knock-down of endogenous L-plastin by siRNA treatment reduced migration of the melanoma cell line IF6. However, in melanoma patients, no correlation existed between L-plastin expression and tumor stages. This implied that additional factors such as phosphorylation of L-plastin may influence its function in tumor cells. To investigate this further, EGFP-tagged wild-type L-plastin (wt-LPL-EGFP) and a mutated, nonphosphorylatable L-plastin protein (5A7A-LPL-EGFP), were expressed in the L-plastin negative melanoma cell line MV3. Biochemical analysis revealed that wt-LPL-EGFP is phosphorylated in MV3 cells while 5A7A-LPL-EGFP is not. Although both wt-LPL-EGFP and 5A7A-LPL-EGFP were targeted to, and promote the formation of, vinculin-containing adhesion sites, static adhesion to either Matrigel or isolated extracellular matrix molecules was neither influenced by expression of wt-LPL-EGFP nor by expression of 5A7A-LPL-EGFP when compared with EGFP expressing control cells. In contrast, haptotactic, but not chemotactic, migration of melanoma cells towards either Matrigel or isolated extracellular matrix molecules was similarly enhanced, if either 5A7A-LPL-EGFP or wt-LPL-EGFP were expressed in MV3 cells. Interestingly, only cells expressing the phosphorylatable wt-LPL-EGFP protein showed enhanced invasion into Matrigel. In line with these findings the in vivo metastatic capacity of mouse B16 melanoma cells correlates with expression and phosphorylation of L-plastin. These data show that an increase in melanoma cell invasiveness requires not only expression but also phosphorylation of L-plastin.

Bacteria communicate with each other via specialized signalling molecules, known as quorum sensing molecules or autoinducers. The Pseudomonas aeruginosa-derived quorum sensing molecule N-(3-oxododecanoyl)-L-homoserine lactone (AHL-12), however, also activates mammalian cells. As shown previously, AHL-12 induced chemotaxis, up-regulated CD11b expression, and enhanced phagocytosis of polymorphonuclear neutrophils (PMN). Circumstantial evidence concurred with a receptor for AHL-12, which so far has been elusive. We investigated the bitter receptor T2R38 as a potential candidate. Although identified as a taste receptor, cells outside the gustatory system express T2R38, for example epithelial cells in the lung. We now detected T2R38 in peripheral blood neutrophils, monocytes and lymphocytes on the cell membrane, but also intracellular. In neutrophils, T2R38 was located in vesicles with characteristics of lipid droplets, and super-resolution microscopy showed a co-localisation with the lipid droplet membrane. Neutrophils take up AHL-12, and it co-localized with T2R38 as seen by laser scan microscopy. Binding of AHL-12 to T2R28 was confirmed by pull-down assays using biotin-coupled AHL-12 as bait. A commercially available antibody to T2R38 inhibited binding of AHL-12 to neutrophils, and this antibody by itself stimulated neutrophils, similarly to AHL-12. In conclusion, our data provide evidence for expression of functional T2R38 on neutrophils, and are compatible with the notion that T2R38 is the receptor for AHL-12 on neutrophils.

Cell adhesion plays an important role in cell-cell contact formation and cell migration. Thus, the assessment of cellular adhesiveness is one important feature when studying cell-mediated immune responses. The interaction of lymphocytes with other cell types such as antigen-presenting cells or vascular-endothelial cells occurs via adhesion molecules including L-selectin, VCAM-1 or ICAM-1. There are principally two mechanisms by which cell adhesion can be enhanced: namely changes in the affinity or avidity of receptor interactions. Conventional plate-based adhesion assays detect both forms. However, they do not permit discrimination between affinity- and avidity-mediated changes in the adhesiveness. Moreover, analysis of cell subpopulations requires cell separation prior to performance of the adhesion assay. Conventional flow-cytometry-based tests make it possible to determine changes in the affinity of integrins at the single cell level. However, they fail to quantify avidity-mediated adhesiveness. Here we describe a novel flow-cytometry-based assay, which allows the detection of both integrin-mediated affinity as well as avidity changes at the single cell level. This opens up the possibility of precisely characterizing the adhesive capacity of subpopulations in heterogeneous cell populations.

The formation of supramolecular activation clusters within the immunological synapse, crucial for sustained signaling and T lymphocyte activation, requires costimulation-dependent reorganization of the actin cytoskeleton. Here we have identified the actin-remodeling protein cofilin as a key player in this process. Cell-permeable peptides that block costimulation-induced cofilin/F-actin interactions in untransformed human T lymphocytes impair receptor capping and immunological synapse formation at the interface between T cells and antigen-presenting cells. As a consequence, T cell activation, as measured by cytokine production and proliferation, is inhibited.

Background Tumor cell migration and metastasis require dynamic rearrangements of the actin cytoskeleton. Interestingly, the F-actin cross-linking and stabilizing protein L-plastin, originally described as a leukocyte specific protein, is aberrantly expressed in several non-hematopoietic malignant tumors. Therefore, it has been discussed as a tumor marker. However, systematic in vivo analyses of the functional relevance of L-plastin for tumor cell metastasis were so far lacking. Methods We investigated the relevance of L-plastin expression and phosphorylation by ectopical expression of L-plastin in human melanoma cells (MV3) and knock-down of endogenous L-plastin in prostate cancer (PC3M). The growth and metastatic potential of tumor cells expressing no L-plastin, phosphorylatable or non-phosphorylatable L-plastin was analyzed in a preclinical mouse model after subcutaneous and intracardial injection of the tumor cells. Results Knock-down of endogenous L-plastin in human prostate carcinoma cells led to reduced tumor cell growth and metastasis. Vice versa, and in line with these findings, ectopic expression of L-plastin in L-plastin negative melanoma cells significantly increased the number of metastases. Strikingly, the metastasis promoting effect of L-plastin was not observed if a non-phosphorylatable L-plastin mutant was expressed. Conclusions Our data provide the first in vivo evidence that expression of L-plastin promotes tumor metastasis and, importantly, that this effect depends on an additionally required phosphorylation of L-plastin. In conclusion, these findings imply that for determining the importance of tumor-associated proteins like L-plastin a characterization of posttranslational modifications is indispensable.

Resident human lamina propria immune cells serve as powerful effectors in host defense. Molecular events associated with the initiation of an intestinal inflammatory response in these cells are largely unknown. Here, we aimed to characterize phenotypic and functional changes induced in these cells at the onset of intestinal inflammation using a human intestinal organ culture model. In this model, healthy human colonic mucosa was depleted of epithelial cells by EDTA treatment. Following loss of the epithelial layer, expression of the inflammatory mediators IL1B, IL6, IL8, IL23A, TNFA, CXCL2, and the surface receptors CD14, TLR2, CD86, CD54 was rapidly induced in resident lamina propria cells in situ as determined by qRT-PCR and immunohistology. Gene microarray analysis of lamina propria cells obtained by laser-capture microdissection provided an overview of global changes in gene expression occurring during the initiation of an intestinal inflammatory response in these cells. Bioinformatic analysis gave insight into signalling pathways mediating this inflammatory response. Furthermore, comparison with published microarray datasets of inflamed mucosa in vivo (ulcerative colitis) revealed a significant overlap of differentially regulated genes underlining the in vivo relevance of the organ culture model. Furthermore, genes never been previously associated with intestinal inflammation were identified using this model. The organ culture model characterized may be useful to study molecular mechanisms underlying the initiation of an intestinal inflammatory response in normal mucosa as well as potential alterations of this response in inflammatory bowel disease.

Tumor-growth is often associated with the expansion of myeloid derived suppressor cells that lead to local or systemic arginine depletion via the enzyme arginase. It is generally assumed that this arginine deficiency induces a global shut-down of T cell activation with ensuing tumor immune escape. While the impact of arginine depletion on polyclonal T cell proliferation and cytokine secretion is well documented, its influence on chemotaxis, cytotoxicity and antigen specific activation of human T cells has not been demonstrated so far. We show here that chemotaxis and early calcium signaling of human T cells are unimpaired in the absence of arginine. We then analyzed CD8(+) T cell activation in a tumor peptide as well as a viral peptide antigen specific system: (i) CD8(+) T cells with specificity against the MART-1aa26-35*A27L tumor antigen expanded with in vitro generated dendritic cells, and (ii) clonal CMV pp65aa495-503 specific T cells and T cells retrovirally transduced with a CMV pp65aa495-503 specific T cell receptor were analyzed. Our data demonstrate that human CD8(+) T cell antigen specific cytotoxicity and perforin secretion are completely preserved in the absence of arginine, while antigen specific proliferation as well as IFN-γ and granzyme B secretion are severely compromised. These novel results highlight the complexity of antigen specific T cell activation and demonstrate that human T cells can preserve important activation-induced effector functions in the context of arginine deficiency.

Dendritic cells (DCs) as professional antigen-presenting cells play an important role in the initiation and modulation of the adaptive immune response. However, their role in the innate immune response against bacterial infections is not completely defined. Here we have analyzed the role of DCs and their impact on the innate anti-bacterial host defense in an experimental infection model of Yersinia enterocolitica (Ye). We used CD11c-diphtheria toxin (DT) mice to deplete DCs prior to severe infection with Ye. DC depletion significantly increased animal survival after Ye infection. The bacterial load in the spleen of DC-depleted mice was significantly lower than that of control mice throughout the infection. DC depletion was accompanied by an increase in the serum levels of CXCL1, G-CSF, IL-1α, and CCL2 and an increase in the numbers of splenic phagocytes. Functionally, splenocytes from DC-depleted mice exhibited an increased bacterial killing capacity compared to splenocytes from control mice. Cellular studies further showed that this was due to an increased production of reactive oxygen species (ROS) by neutrophils. Adoptive transfer of neutrophils from DC-depleted mice into control mice prior to Ye infection reduced the bacterial load to the level of Ye-infected DC-depleted mice, suggesting that the increased number of phagocytes with additional ROS production account for the decreased bacterial load. Furthermore, after incubation with serum from DC-depleted mice splenocytes from control mice increased their bacterial killing capacity, most likely due to enhanced ROS production by neutrophils, indicating that serum factors from DC-depleted mice account for this effect. In summary, we could show that DC depletion triggers phagocyte accumulation in the spleen and enhances their anti-bacterial killing capacity upon bacterial infection., Author Summary Dendritic cells (DCs) are professional antigen-presenting cells playing a crucial role in the initiation of T-cell responses to combat infection. DCs adapt their immune response according to the type of pathogen. For example, in response to intracellular bacteria, DCs produce IL-12, thereby initiating Th1 polarization, whereas in response to extracellular parasites or extracellular bacteria, DCs instruct Th2 or Th17 polarization, respectively. Nevertheless, their role in innate immunity is less well understood. To address this, we studied the role of DCs upon infection with the Gram-negative enteropathogenic bacteria Yersinia enterocolitica (Ye) and used a mouse model to deplete DCs. We found that DCs have an unexpected role during severe infection as depletion of these cells resulted in better outcome of infection as well as less bacterial load. We also found that DC depletion increased the number of phagocytes with improved anti-bacterial capacity in the spleen. Our study provides new insights into the role of DCs in innate immune response upon bacterial infection and points towards a complex interaction between DCs and phagocyte homeostasis. DC alteration during infection might also be an interesting target for immunotherapy in the future to guide the outcome of infection.

T-cell recognition of peptide-MHC complexes on APCs requires cell-cell interactions. The molecular events leading to T-cell activation have been extensively investigated, but the underlying physical binding forces between T-cells and APCs are largely unknown. We used single cell force spectroscopy for quantitation of interaction forces between T-cells and APCs presenting a tolerogenic peptide derived from myelin basic protein. When T-cells were brought into contact with peptide-loaded APCs, interaction forces increased with time from about 0.5 nN after 10 seconds interaction to about 15 nN after 30 minutes. In the absence of antigen, or when ICAM-1-negative APC were used, no increase in binding forces was observed. The temporal development of interaction forces correlated with the kinetics of immune synapse formation, as determined by LFA-1 and TCR enrichment at the interface of T-cell/APC conjugates using high throughput multispectral imaging flow cytometry. Together, these results suggest that ICAM-1/LFA-1 redistribution to the contact area is mainly responsible for development of strong interaction forces. High forces will keep T-cells and APCs in tight contact, thereby providing a platform for optimal interaction between TCRs and peptide-MHC complexes.

Oxidative stress leads to T-cell hyporesponsiveness or death. The actin-binding protein cofilin is oxidized during oxidative stress, which provokes a stiff actin cytoskeleton and T-cell hyporesponsiveness. Here, we show that long-term oxidative stress leads to translocation of cofilin into the mitochondria and necrotic-like programmed cell death (PCD) in human T cells. Notably, cofilin mutants that functionally mimic oxidation by a single mutation at oxidation-sensitive cysteins (Cys-39 or Cys-80) predominately localize within the mitochondria. The expression of these mutants alone ultimately leads to necrotic-like PCD in T cells. Accordingly, cofilin knockdown partially protects T cells from the fatal effects of long-term oxidative stress. Thus, we introduce the oxidation and mitochondrial localization of cofilin as the checkpoint for necrotic-like PCD upon oxidative stress as it occurs, for example, in tumor environments.

Formation of immune synapses (IS) between T cells and APC requires multiple rearrangements in the actin cytoskeleton and selective receptor accumulation in supramolecular activation clusters (SMAC). The inner cluster (central SMAC) contains the TCR/CD3 complex. The outer cluster (peripheral SMAC) contains the integrin LFA-1 and Talin. Molecular mechanisms selectively stabilizing receptors in the IS remained largely unknown. Here, we demonstrate that sustained LFA-1 clustering in the IS is a consequence of the combined activities of the actin-bundling protein L-plastin (LPL) and calmodulin. Thus, upon antigen-recognition of T cells, LPL accumulated predominantly in the peripheral SMAC. siRNA-mediated knock-down of LPL led to a failure of LFA-1 and Talin redistribution – however, not TCR/CD3 relocalization – into the IS. As a result of this LPL knock-down, the T-cell/APC interface became smaller over time and T-cell proliferation was inhibited. Importantly, binding of calmodulin to LPL was required for the maintenance of LPL in the IS and consequently inhibition of calmodulin also prevented stable accumulation of LFA-1 and Talin, but not CD3, in the IS.

T cells infiltrate peripheral tissues to execute immunosurveillance and effector functions. For this purpose, T cells first migrate on the two-dimensional (2D) surface of endothelial cells to undergo transendothelial migration. Then they change their mode of movement to undergo migration within the three-dimensional (3D)-extracellular matrix of the infiltrated tissue. As yet, no molecular mechanisms are known, which control migration exclusively in either 2D or 3D environments. Here, we describe a signalling module that controls T-cell chemotaxis specifically in 3D environments. In chemotaxing T cells, Ras activity is spatially restricted to the lamellipodium. There, Ras initiates activation of MEK, which in turn inhibits LIM-kinase 1 activity, thereby allowing dephosphorylation of the F-actin-remodelling protein cofilin. Interference with this MEK-cofilin module by either inhibition of MEK or by knockdown of cofilin reduces speed and directionality of chemotactic migration in 3D-extracellular matrices, but not on 2D substrates. This MEK-cofilin module may have an important function in the tissue positioning of T cells during an immune response.

H(2)O(2) acts as a signaling molecule by oxidizing critical thiol groups on redox-regulated target proteins. To explain the efficiency and selectivity of H(2)O(2)-based signaling, it has been proposed that oxidation of target proteins may be facilitated by H(2)O(2)-scavenging peroxidases. Recently, a peroxidase-based protein oxidation relay has been identified in yeast, namely the oxidation of the transcription factor Yap1 by the peroxidase Orp1. It has remained unclear whether the protein oxidase function of Orp1 is a singular adaptation or whether it may represent a more general principle. Here we show that Orp1 is in fact not restricted to oxidizing Yap1 but can also form a highly efficient redox relay with the oxidant target protein roGFP (redox-sensitive green fluorescent protein) in mammalian cells. Orp1 mediates near quantitative oxidation of roGFP2 by H(2)O(2), and the Orp1-roGFP2 redox relay effectively converts physiological H(2)O(2) signals into measurable fluorescent signals in living cells. Furthermore, the oxidant relay phenomenon is not restricted to Orp1 as the mammalian peroxidase Gpx4 also mediates oxidation of proximal roGFP2 in living cells. Together, these findings support the concept that certain peroxidases harbor an intrinsic and powerful capacity to act as H(2)O(2)-dependent protein thiol oxidases when they are recruited into proximity of oxidizable target proteins.

Cardiac troponins provide excellent risk stratification in unstable angina (UA), but no reliable markers are available in troponin-negative patients. Beta2-integrin mediated T cell recruitment plays a pivotal role in coronary atherosclerotic plaque rupture. The present study investigates beta2-integrin activation on T cell subsets as a risk marker in UA. Functional activation (affinity/avidity) of beta2-integrins on T cells was measured using a flow cytometry-based whole blood assay in 87 patients with UA. Beta2-integrin activation was significantly higher in patients with severe coronary artery disease (sC) and myocardial infarction (MI) compared to patients with no/minimal coronary atherosclerosis (no/mC), irrespective of troponin status. Adjusted for cardiovascular risk factors, medication, left ventricular function, MI at enrollment and high sensitivity C-reactive protein (hsCRP), beta2-integrin activation was independently associated with incidence of revascularization, hospitalization and all major cardiovascular events during 9 months of follow-up after index investigation. The highest prognostic value of beta2-integrin activation was seen in troponin-and hsCRP-negative patients. Quantitative assessment of T cell beta2-integrin activation allows to identify high risk patients with UA and sC without established MI; furthermore, it is associated with incidence of future cardiovascular events independent of conventional risk factors (troponin, hsCRP).

T cell activation requires costimulation of TCR/CD3 plus accessory receptors (e.g. CD28). A hallmark of costimulation is the dynamic reorganization of the actin cytoskeleton, important for receptor polarization in the immunological synapse. The classical model of T cell costimulation was challenged by the detection of superagonistic anti-CD28 antibodies. These induce T cell proliferation and--as demonstrated here--production of IFN-gamma, CD25 and CD69 even in the absence of TCR/CD3 coligation. Here, we analyzed whether superagonistic CD28 stimulation induces costimulatory signaling events. Costimulation leads to phosphorylation of the actin-bundling protein L-plastin and dephosphorylation of the actin-reorganizing protein cofilin. Cofilin binds to F-actin only in its dephosphorylated form. Binding of cofilin to F-actin leads to depolymerization or severing of F-actin. The latter ends up in smaller F-actin fragments, which can be elongated at the free barbed ends. This results in enhanced actin polymerization. Dephosphorylation of cofilin requires activation of Ras and PI3Kinase. Interestingly, superagonistic CD28 stimulation activates human peripheral blood T cells independently of Ras and PI3Kinase. Accordingly, it does not lead to cofilin dephosphorylation and receptor polarization. Likewise, L-plastin is not phosphorylated. Thus, superagonistic CD28 stimulation does not mimic costimulation. Instead, it leads to a Ras/PI3Kinase/cofilin-independent state of "unpolarized T cell activation".

Lipid rafts and the formation of an immunological synapse are crucial for T-cell activation. Binding of cholera toxin B subunit (CTB) to ganglioside GM1 is a marker to identify lipid rafts. Primary human T cells were isolated from healthy donors and were stimulated with superantigen staphylococcus enterotoxin B (SEB) and stained with cholera toxin B-fluorescein isothiocyanate (CTB-FITC). An optimized staining procedure is required to stain lipid rafts exclusively on the cell surface. Unstimulated T cells show a few CTB binding spots on the cell surface. The size and number of CTB-binding lipid rafts are strongly upregulated during T-cell activation in SEB-stimulated CD4(+) T cells. However, our data show that the specificity of CTB for GM1 ganglioside is limited, because the binding capacity is partly resistant to inhibition of ganglioside synthesis and sensitive to trypsin digestion. Our results indicate that the binding of FITC-labeled CTB can be divided into at least three different categories: a specific binding of CTB to ganglioside GM1, a nonspecific binding of CTB probably to glycosylated surface proteins and a nonspecific binding of FITC to the cell surface.

Adhesion of leukocytes is an early step in the formation of adaptive or innate immunity. In chronic inflammatory pathologies like atherosclerosis, regulation of adhesiveness is pivotal for the accumulation of leukocytes within the vessel wall. Therefore, the quantification of adhesion is crucial for the understanding and monitoring of immune responses in patients. However, so far, functional analysis of leukocyte adhesion has been time consuming and required prior purification of cell populations from peripheral blood. This reduced the number of samples and cell populations that could be analysed from limited patient material. Here, we introduce a novel method involving rapid quantification of integrin-mediated leukocyte adhesion in human whole blood using flow cytometry. The quantification relies on soluble multivalent immunocomplexes and is thus called “ligand-complex-based adhesion assay” (LC-AA). LC-AA evaluates both integrin affinity and avidity in T-cells, NK-cells and monocytes from as little as 20 μl of whole blood. In marked contrast to T-cells and NK-cells, unstimulated monocytes show non-blockable background binding of the complexes. Therefore, for this subset only, the stimulation-induced integrin activation is measurable. With the LC-AA, for the first time, measurement of adhesiveness of extremely rare cell populations like CD34+ peripheral blood stem cells can be assessed in the absence of prior purification steps. Finally, the small blood volumes needed for adhesion analysis with the LC-AA allow the evaluation of multiple cell subpopulations in large sample collectives, e.g. required in clinical studies.

The activity of cofilin, an actin-remodeling protein, is required for T lymphocyte activation with regard to formation of the immunological synapse, cytokine production, and proliferation. In unstimulated T PBL (PB-T), cofilin is present in its Ser3-phosphorylated inactive form. Costimulation of TCR/CD3 and CD28 induces dephosphorylation and, thus, activation of cofilin. In this study we characterized the signaling cascades leading to cofilin activation in untransformed human PB-T. We show that a Ras-PI3K cascade regulates dephosphorylation of cofilin in PB-T. The GTPase Ras is a central mediator of this pathway; transient expression of an activated form of H-Ras in PB-T triggered the dephosphorylation of cofilin. Inhibition of either MAPK/ERK kinase or PI3K blocked both Ras-induced and costimulation-induced cofilin dephosphorylation in PB-T, showing that the combined activities of both signaling proteins are required to activate cofilin. That Ras functions as a central regulator of cofilin dephosphorylation after costimulation through CD3 × CD28 was finally proven by transient expression of a dominant negative form of H-Ras in primary human PB-T. It clearly inhibited costimulation-induced cofilin dephosphorylation, and likewise, activation of PI3K was diminished. Our data, in addition, demonstrate that regarding the downstream effectors of Ras, a clear difference exists between untransformed human PB-T and the T lymphoma line Jurkat. Thus, in PB-T the Ras signaling cascade is able to activate PI3K, whereas in Jurkat cells this is not the case. In addition to the insights into the regulation of cofilin, this finding discloses a to date unrecognized possibility of PI3K activation in T lymphocytes.

Dynamic rearrangements of the actin cytoskeleton are crucial for the function of numerous cellular elements including T lymphocytes. They are required for migration of T lymphocytes through the body to scan for the presence of antigens, as well as for the formation and stabilization of the immunological synapse at the interface between antigen-presenting cells and T lymphocytes. Supramolecular activation clusters within the immunological synapse play an important role for the initiation of T cell responses and for the execution of T cell effector functions. In addition to the T cell receptor/CD3 induced actin nucleation via Wasp/Arp2/3-activation, signals through accessory receptors of the T cell (i.e., costimulation) regulate actin cytoskeletal dynamics. In this regard, the actin-binding proteins cofilin and L-plastin represent prominent candidates linking accessory receptor stimulation to the rearrangement of the actin cytoskeleton. Cofilin enhances actin polymerization via its actin-severing activity, and as a long-lasting effect, cofilin generates novel actin monomers through F-actin depolymerization. L-plastin stabilizes acin filament structures by means of its actin-bundling activity.

Selenium appears to fall into a category of substances that have an effect on growth, and in a not completely explained fashion is related to sulfur metabolism (Desai et al., 1964). Congenital abnormalities (monstrosities) are known to occur in chick embryos or in chicks hatched from eggs laid by hens fed toxic amounts of selenium or grains containing these compounds (Tully and Franke, 1935). In contrast there is evidence that trace amounts of selenium are involved in the normal growth process in plants (Trelease and Beath, 1949), microorganisms (Pinsent, 1954) and fowl (Nesheim and Scott, 1958; Zalkin et al., 1960). General knowledge and understanding of the specific relationship between selenium and growth are limited. The chick embryo was selected as the experimental animal in the studies on growth and development since it is during the early embryo stages that a relatively rapid and profound developmental activity occurs when the plans…