Immune monitoring design within the developmental pipeline for an immunotherapeutic or preventive vaccine

Immunotherapy is defined as the treatment of disease by inducing, enhancing, or suppressing an immune response, whereas preventive vaccination is intended to prevent the development of diseases in healthy subjects. Most successful prophylactic vaccines rely on the induction of high titers of neutralizing antibodies. It is generally thought that therapeutic vaccination requires induction of robust T-cell mediated immunity. The diverse array of potential or already in use immunotherapeutic and preventive agents all share the commonality of stimulating the immune system. Hence, measuring those vaccination-induced immune responses gives the earliest indication of vaccine take and its immune modulating effects.

Quantitative multiparameter assays to measure the effect of adjuvants on human antigen-specific CD8 T-cell responses.

Large numbers and functionally competent T cells are required to protect from diseases for which antibody-based vaccines have consistently failed (1), which is the case for many chronic viral infections and solid tumors. Therefore, therapeutic vaccines aim at the induction of strong antigen-specific T-cell responses. Novel adjuvants have considerably improved the capacity of synthetic vaccines to activate T cells, but more research is necessary to identify optimal compositions of potent vaccine formulations. Consequently, there is a great need to develop accurate methods for the efficient identification of antigen-specific T cells and the assessment of their functional characteristics directly ex vivo. In this regard, hundreds of clinical vaccination trials have been implemented during the last 15 years, and monitoring techniques become more and more standardized.

Phosphate Deficiency Induces the Jasmonate Pathway and Enhances Resistance to Insect Herbivory.

During their life cycle, plants are typically confronted by simultaneous biotic and abiotic stresses. Low inorganic phosphate (Pi) is one of the most common nutrient deficiencies limiting plant growth in natural and agricultural ecosystems, while insect herbivory accounts for major losses in plant productivity and impacts ecological and evolutionary changes in plant populations. Here, we report that plants experiencing Pi deficiency induce the jasmonic acid (JA) pathway and enhance their defense against insect herbivory. Pi-deficient Arabidopsis (Arabidopsis thaliana) showed enhanced synthesis of JA and the bioactive conjugate JA-isoleucine, as well as activation of the JA signaling pathway, in both shoots and roots of wild-type plants and in shoots of the Pi-deficient mutant pho1 The kinetics of the induction of the JA signaling pathway by Pi deficiency was influenced by PHOSPHATE STARVATION RESPONSE1, the main transcription factor regulating the expression of Pi starvation-induced genes. Phenotypes of the pho1 mutant typically associated with Pi deficiency, such as high shoot anthocyanin levels and poor shoot growth, were significantly attenuated by blocking the JA biosynthesis or signaling pathway. Wounded pho1 leaves hyperaccumulated JA/JA-isoleucine in comparison with the wild type. The pho1 mutant also showed an increased resistance against the generalist herbivore Spodoptera littoralis that was attenuated in JA biosynthesis and signaling mutants. Pi deficiency also triggered increased resistance to S. littoralis in wild-type Arabidopsis as well as tomato (Solanum lycopersicum) and Nicotiana benthamiana, revealing that the link between Pi deficiency and enhanced herbivory resistance is conserved in a diversity of plants, including crops.

Cultured human fetal astrocytes can be induced by interferon-gamma to express HLA-DR.

Interferon-gamma (IFN-gamma) modulates the expression of Class II major histocompatibility antigens (MHC), thus providing a potential regulatory mechanism for local immune reactivity in the context of MHC-restricted antigen presentation. Within the central nervous system (CNS), the expression of MHC Class II antigens has been demonstrated on human reactive astrocytes and glioma cells. In order to investigate the modulation of HLA-DR on normal astrocytes, two cell lines were grown from a 20-week-old fetal brain. In situ none of the fetal brain cells expressed HLA-DR as determined by immunohistology on frozen tissue sections. The two cell lines, FB I and FB II, expressed GFAP indicating their astrocytic origin. FB I was HLA-DR negative at the first tissue culture passages, but could be induced to express HLA-DR when treated with 500 U/ml IFN-gamma. FB II was spontaneously HLA-DR positive in the early passages, lost the expression of this antigen after 11 passages and could also be induced to express HLA-DR by IFN-gamma. The induction of HLA-DR expression was demonstrated both by a binding RIA and by immunoprecipitation using a monoclonal antibody (MAB) directed against a monomorphic determinant of HLA-DR. The HLA-DR alloantigens were determined on FB II cells after IFN-gamma treatment, by immunofluorescence and by cytotoxicity assays, and were shown to be DR4, DR6, Drw52, DRw53 and DQwl. These results show that human fetal astrocytes can be induced to express HLA-DR by IFN-gamma in vitro and support the concept that astrocytes may function as antigen-presenting cells.

Role of intracellular signaling pathways activated in fibroblasts in response to lipoproteins

Summary The best described physiological function of low-density lipoproteins (LDL) is to transport cholesterol to target tissues. LDL deliver their cholesterol cargo to cells following their interaction with the LDL receptor. LDL, when their vascular concentrations increase, have also been implicated in pathologies such as atherosclerosis. Among the cell types that are found in blood vessels, endothelial and smooth muscle cells have dominated cellular research on atherosclerotic mechanisms and LDL activation of signaling pathways, while very little is known about adventitial fibroblast activation caused by elevated lipoprotein levels. Since fibroblasts participate in wound repair and since it has recently been recognized that fibroblasts may play pivotal roles in vascular remodeling and repair of injury, we assessed whether lipoproteins affect fibroblast function. We have found that LDL specifically mediate the activation of a class of mitogen-activated protein kinases (MAPKs): the p38 MAPKs. The activation of this pathway in turn modulates cell shape by promoting lamellipodia formation and extensive cell spreading. This is of particular interest because it provides a mechanism by which LDL can promote wound healing or vessel wall remodeling as observed during the development of atherosclerosis. In order to understand the molecular mechanisms by which LDL induce p38 activation we searched for the component in the LDL particle responsible for the induction of this pathway. We found that cholesterol is the major component of lipoprotein particles that mediates their ability to stimulate the p38 MAPK pathway. Furthermore, we investigated the cellular mechanisms underlying the ability of LDL to induce cell shape changes and whether this could participate in wound repair. Our recent data demonstrates that the capacity of LDL to induce fibroblast spreading relies on their ability to stimulate IL-8 secretion, which in turn leads to accelerated wound healing. LDL-induced IL-8 production and subsequent wound closure are impaired upon inhibition of the p38 MAPK pathway indicating that the LDL-induced spreading and accelerated wound sealing rely on the ability of LDL to stimulate IL-8 secretion in a p38 MAPK-dependent manner. Therefore, regulation of fibroblast shape and migration by lipoproteins may be relevant to atherosclerosis that is characterized by increased LDL-cholesterol levels, IL-8 production and extensive remodeling of the vessel wall. Résumé: La fonction physiologique des lipoprotéines à faible densité (LDL) la mieux décrite est celle du transport du cholestérol aux tissus cibles. Les LDL livrent leur cargaison de cholestérol aux cellules après leur interaction avec le récepteur au LDL. Une concentration vasculaire des LDL augmenté est également impliquée dans le développement de l'athérosclérose. Parmi les types de cellule présents dans les vaisseaux sanguins, les cellules endothéliales et les cellules du muscle lisse ont dominé la recherche cellulaire sur les mécanismes athérosclérotiques et sur l'activation par les LDL des voies de signalisation intracellulaire. A l'inverse peu de choses sont connues sur l'activation des fibroblastes de l'adventice par les lipoprotéines. Puisqu'il a été récemment reconnu que les fibroblastes peuvent jouer un rôle central dans la remodélisation vasculaire et la réparation tissulaire, nous avons étudié si les lipoprotéines affectent la fonction des fibroblastes. Nous avons constaté que les LDL activent spécifiquement une classe de protéines kinases: les p38 MAPK (mitogen-activated protein kinases). L'activation de cette voie module à son tour la forme de la cellule en favorisant la formation de lamellipodes et l'agrandissement des cellules. Cela a un intérêt particulier car il fournit un mécanisme par lequel les LDL peuvent promouvoir la cicatrisation ou la remodélisation des parois vasculaires comme observés lors du développement de l'athérosclérose. Pour comprendre les mécanismes moléculaires par lesquels les LDL provoquent l'activation des p38 MAPK, nous avons cherché à identifier les composants dans la particule de LDL responsables de l'induction de cette voie. Nous avons constaté que le cholestérol est l'élément principal des particules de lipoprotéine qui contrôle leur capacité à stimuler la voie des p38 MAPK. En outre, nous avons examiné les mécanismes cellulaires responsables de la capacité des LDL à induire des changements dans la forme des cellules. Nos données récentes démontrent que la capacité des LDL à induire l'agrandissement des cellules, ainsi que leur aptitude à favoriser la cicatrisation, reposant sur leur capacité à stimuler la sécrétiond'IL-8. La production d'IL-8 induite par les LDL est bloquée par l'inhibition de la voie p38 MAPK, ce qui indique que l'étalement des cellules induit par les LDL ainsi que l'accélération de la cicatrisation sont liés à la capacité des LDL à stimuler la sécrétion d'IL8 via l'activation des p38 MAPK. La régulation de la forme et de la migration des fibroblastes par les lipoprotéines peuvent donc participer au développement de l'athérosclérose qui est caractérisée par l'augmentation des niveaux de production de LDL-cholestérol et d'IL-8 ainsi que par une remodélisation augmentée de la paroi du vaisseau.

Flagellin promotes myeloid differentiation factor 88-dependent development of Th2-type response.

Activation of dendritic cells (DC) by microbial products via Toll-like receptors (TLR) is instrumental in the induction of immunity. In particular, TLR signaling plays a major role in the instruction of Th1 responses. The development of Th2 responses has been proposed to be independent of the adapter molecule myeloid differentiation factor 88 (MyD88) involved in signal transduction by TLRs. In this study we show that flagellin, the bacterial stimulus for TLR5, drives MyD88-dependent Th2-type immunity in mice. Flagellin promotes the secretion of IL-4 and IL-13 by Ag-specific CD4(+) T cells as well as IgG1 responses. The Th2-biased responses are associated with the maturation of DCs, which are shown to express TLR5. Flagellin-mediated DC activation requires MyD88 and induces NF-kappaB-dependent transcription and the production of low levels of proinflammatory cytokines. In addition, the flagellin-specific response is characterized by the lack of secretion of the Th1-promoting cytokine IL-12 p70. In conclusion, this study suggests that flagellin and, more generally, TLR ligands can control Th2 responses in a MyD88-dependent manner.

Malt1 protease inactivation efficiently dampens immune responses but causes spontaneous autoimmunity.

The protease activity of the paracaspase Malt1 has recently gained interest as a drug target for immunomodulation and the treatment of diffuse large B-cell lymphomas. To address the consequences of Malt1 protease inactivation on the immune response in vivo, we generated knock-in mice expressing a catalytically inactive C472A mutant of Malt1 that conserves its scaffold function. Like Malt1-deficient mice, knock-in mice had strong defects in the activation of lymphocytes, NK and dendritic cells, and the development of B1 and marginal zone B cells and were completely protected against the induction of autoimmune encephalomyelitis. Malt1 inactivation also protected the mice from experimental induction of colitis. However, Malt1 knock-in mice but not Malt1-deficient mice spontaneously developed signs of autoimmune gastritis that correlated with an absence of Treg cells, an accumulation of T cells with an activated phenotype and high serum levels of IgE and IgG1. Thus, removal of the enzymatic activity of Malt1 efficiently dampens the immune response, but favors autoimmunity through impaired Treg development, which could be relevant for therapeutic Malt1-targeting strategies.

Ectodysplasin A (EDA) - EDA receptor signalling and its pharmacological modulation.

The TNF family ligand ectodysplasin A (EDA) regulates the induction, morphogenesis and/or maintenance of skin-derived structures such as teeth, hair, sweat glands and several other glands. Deficiencies in the EDA - EDA receptor (EDAR) signalling pathway cause hypohidrotic ectodermal dysplasia (HED). This syndrome is characterized by the absence or malformation of several skin-derived appendages resulting in hypotrychosis, hypodontia, heat-intolerance, dry skin and dry eyes, susceptibility to airways infections and crusting of various secretions. The EDA-EDAR system is an important effector of canonical Wnt signalling in developing skin appendages. It functions by stimulating NF-κB-mediated transcription of effectors or inhibitors of the Wnt, Sonic hedgehog (SHH), fibroblast growth factor (FGF) and transforming growth factor beta (TGFβ) pathways that regulate interactions within or between epithelial and mesenchymal cells and tissues. In animal models of Eda-deficiency, soluble EDAR agonists can precisely correct clinically relevant symptoms with low side effects even at high agonist doses, indicating that efficient negative feedback signals occur in treated tissues. Hijacking of the placental antibody transport system can help deliver active molecules to developing foetuses in a timely manner. EDAR agonists may serve to treat certain forms of ectodermal dysplasia.