Tolerogenic dendritic cells and their role in induction of immune tolerance

Les cellules dendritiques sont des cellules du système immunitaire qui permettent d'instruire les lymphocytes T, autres cellules de ce système, pour mettre en place une réponse immunitaire adaptée afin de combattre et vaincre une infection. Ces cellules dendritiques vont reconnaître des motifs spécifiquement exprimés par des pathogènes par l'intermédiaire de récepteurs exprimés à leur surface. En détectant ces molécules, elles vont s'activer et subir diverses modifications pour pouvoir activer les lymphocytes T. Elles vont alors interagir avec les lymphocytes Τ et transférer les informations nécessaires pour que ces cellules s'activent à leur tour et produisent différentes protéines de façon à éliminer le pathogène. En fonction du type de pathogène, les informations transférées entre les cellules dendritiques et les lymphocytes seront différentes de manière à produire la réponse immunitaire la mieux adaptée pour supprimer l'élément infectieux. Dans le corps, les cellules dendritiques circulent continuellement afin de détecter les éléments étrangers. Quand elles reconnaissent une protéine étrangère, elles la phagocytent, c'est-à-dire qu'elles la mangent afin de pouvoir la présenter aux lymphocytes T. Mais quand elles phagocytent un élément étranger, elles peuvent également prendre des éléments du soi, comme par exemple quand elles phagocytent une cellule infectée par un virus. Les cellules dendritiques doivent alors être capables de différentier les molécules du soi et du non-soi de façon à ne pas induire une réponse en présentant un antigène du soi aux lymphocytes T. D'autant plus que lors de leur développement, les lymphocytes Τ qui sont capables de reconnaître le soi sont éliminés mais ce système n'est pas parfait et donc certains lymphocytes Τ auto-reactifs peuvent se trouver dans le corps. Il existe ainsi d'autres mécanismes en périphérie du site de développement pour inhiber ces lymphocytes Τ auto-reactifs. Ce sont les mécanismes de tolérance. Quand les lymphocytes Τ induisent une réponse aux antigènes du soi, cela résulte à des maladies auto-immunes. Dans mon projet de recherche, nous avons travaillé avec des lignées de cellules dendritiques, c'est-à-dire des cellules dendritiques semblables à celles que l'on peut trouver in vivo mais qui sont immortalisées, elles peuvent donc être cultiver et manipuler in vitro. Nous avons génétiquement modifiées ces lignées cellulaires pour qu'elles expriment des molécules immunosuppressives afin d'étudier comment induire une tolérance immunitaire, c'est-à-dire si l'expression de ces molécules permet d'éviter de générer une réponse immunitaire. Pour cela, nous avons utilisé des modèles murins de tumeurs et de maladies auto-immunes. Nous avons démontré que ces lignées de cellules dendritiques peuvent être un grand outil de recherche pour étudier les bénéfices de différentes molécules immuno-modulatrices afin d'induire une tolérance immunitaire à différents antigènes. - Les cellules dendritiques sont responsables de l'induction des réponses immunitaires adaptatives. Suite à une infection microbienne, les cellules dendritiques s'activent, elles induisent l'expression de molécules de costimulation à leur surface, sécrètent des cytokines et induisent la différentiation des cellules Τ effectrices et mémoires. De plus, les cellules dendritiques ont un rôle important dans l'induction et la maintenance de la tolérance immunitaire au niveau du thymus et en périphérie, en induisant l'anergie, la délétion ou la conversion des cellules Τ naïves en cellules régulatrices. Dans notre groupe, une nouvelle lignée de cellules dendritiques appelée MuTu a été crée par la culture de cellules dendritiques tumorales isolées à partir d'une rate d'une souris transgénique, dans laquelle l'expression de l'oncogène SV40 et du GFP sont sous le contrôle du promoteur CD1 le, et sont ainsi spécifiquement exprimés dans les cellules dendritiques. Ces nouvelles lignées appartiennent au sous-type des cellules dendritiques conventionnelles exprimant CD8a. Elles ont conservé leur capacité d'augmenter l'expression des marqueurs de costimulation à leur surface ainsi que le production de cytokines en réponse à des ligands des récepteurs Toll, ainsi que leur capacité à présenter des antigènes associés aux molécules du complexe majeur d'histocompatibilité (CMH) de classe I ou II pour activer la prolifération et la différentiation des lymphocytes T. En utilisant un système de transduction de lentivirus de seconde génération, ces nouvelles lignées de cellules dendritiques ont été génétiquement modifiées pour sur-exprimer des molécules immunosuppressives (IL-10, TGFP latent, TGFp actif, Activin A, Arginase 1, IDO, B7DC et CTLA4). Ces lignées permettent d'étudier de manière reproductible le rôle de ces molécules potentiellement tolérogènes sur les réponses immunitaires in vitro et in vivo. Ces lignées potentiellement tolérogènes ont été testées, tout d'abord, in vitro, pour leur capacité à inhiber l'activation des cellules dendritiques, à bloquer la prolifération des cellules Τ ou à modifier leur polarisation. Nos résultats démontrent qu'en réponse à une stimulation, la sur-expression des molécules costimulatrices et la sécrétion de molécules pro- inflammatoires est réduite quand les cellules dendritiques sur-expriment l'IL-10. La sur¬expression de TGFp sous sa forme active induit le développement de cellules régulatrices CD4+ CD25+ Foxp3+ et bloque la réponse CD8 cytotoxique tandis que la sur-expression de CTLA4 à la surface des cellules dendritiques inhibe une réponse Thl et induit des lymphocytes Τ anergiques. Ces lignées ont également été utilisées pour étudier l'induction de tolérance in vivo. Tout d'abord, nous avons étudié l'induction de tolérance dans un modèle de développement de tumeurs. En effet, quand les lignées tumorales sont transférées dans les lignées de souris C57BL/6, elles sont reconnues comme du non-soi du à l'expression de l'oncogène SV40 et du GFP et sont éliminées. Ce mécanisme d'élimination a été étudié en utilisant une lignée de cellules dendritiques modifiée pour exprimer la luciférase et qui a permis de suivre le développement des tumeurs par de l'imagerie in vivo dans des animaux vivants. Ces lignées de cellules dendritiques MuTu sont éliminées dans la souris C57BL/6 par les lymphocytes CD8 et l'action cytotoxique de la perforine. Après plusieurs injections, les cellules dendritiques sur-exprimant CTLA4 ou l'actif TGFp peuvent casser cette réponse immunitaire inhérente aux antigènes de la lignée et induire le développement de la tumeur dans la souris C57BL/6. Le développement tumoral a pu être suivi en mesurant la bioluminescence émise par des cellules dendritiques modifiées pour exprimer à la fois l'actif TGFp et la luciférase. Ces tumeurs ont pu se développer grâce à la mise en place d'un microenvironnement suppressif pour échapper à l'immunité en recrutant des cellules myéloïde suppressives, des lymphocytes CD4 régulateurs et en induisant l'expression d'une molécule inhibitrice PD-1 à la surface des lymphocytes CD8 infiltrant la tumeur. Dans un deuxième temps, ces lignées tolérogènes ont également été testées dans un modèle murin de maladies auto-immunes, appelé l'encéphalomyélite auto-immune expérimental (EAE), qui est un modèle pour la sclérose en plaques. L'EAE a été induite dans la souris par le transfert de cellules de ganglions prélevées d'une souris donneuse préalablement immunisée avec une protéine du système nerveux central, la glycoprotéine myéline oligodendrocyte (MOG) émulsifiée dans de l'adjuvant complet de Freund. La vaccination des souris donneuses et receveuses avec les cellules sur-exprimant l'actif TGFP préalablement chargées avec la protéine MOG bloque l'induction de l'EAE. Nous sommes actuellement en train de définir les mécanismes qui permettent de protéger la souris du développement de la maladie auto-immune. Dans cette étude, nous avons ainsi démontré la possibilité d'induire la tolérance in vivo et in vitro à différents antigènes en utilisant nos nouvelles lignées de cellules dendritiques et en les modifiant pour exprimer des molécules immunosuppressives. En conséquence, ces nouvelles lignées de cellules dendritiques représentent un outil pour explorer les bénéfices de différentes molécules ayant des propriétés immuno-modulatrices pour manipuler le système immunitaire vers un phénotype tolérogène. - Dendritic cells (DC) are widely recognized as potent inducers of the adaptive immune responses. Importantly, after microbial infections, DC become activated, induce co- stimulation, secrete cytokines and induce effector and memory Τ cells. DC furthermore play an important role in inducing and maintaining central and peripheral tolerance by inducing anergy, deletion or commitment of antigen-specific naïve Τ cells into regulatory Τ cells. In our group, stable MuTu DC lines were generated by culture of splenic DC tumors from transgenic mice expressing the SV40 large Τ oncogene and the GFP under DC-specific CDllc promoter. These transformed DC belong to the CD8a+ conventional DC subtype and have fully conserved their capacity to upregulate co-stimulatory markers and produce cytokines after activation with Toll Like Receptors-ligands, and to present Major Histocompatibility class-I or MHCII-restricted antigens to activate Τ cell expansion and differentiation. Using a second- generation lentiviral transduction system, these newly developed MuTu DC lines were genetically modified to overexpress immunosuppressive molecules (IL-10, latent TGFp, active TGFp, Activin A, Arginase 1, IDO, B7DC and CTLA4). This allows to reproducibly investigate the role of these potentially tolerogenic molecules on in vitro and in vivo immune responses. These potentially tolerogenic DC were tested in vitro for their ability to inhibit DC activation, to prevent Τ cell proliferation and to modify Τ cell polarization. Our results show that the upregulation of costimulatory molecules and the secretion of pro-inflammatory cytokines were reduced upon stimulation of DC overexpressing IL-10. The overexpression of active TGFP induced the development of CD4+ CD25+ Foxp3+ regulatory Τ cells and inhibited the cytotoxic CD8 Τ cell response as shown by using the OT-II Τ cell system whereas the surface expression of CTLA-4 on DC prevented the Thl response and prompted an anergic antigen-specific Τ cell response. These MuTu DC lines were also used in vivo in order to study the induction of tolerance. First we addressed the induction of tolerance in a model of tumorogenesis. The adoptively transferred tumor cell lines were cleared in C57BL/6 mice due to the foreign expression of SV40 LargeT and GFP. The mechanism of clearance of MuTu DC line into C57BL/6 mice was investigated by using luciferase-expressing DC line. These DC line allowed to follow, by in vivo imaging, the tumor development in living animals and determined that MuTu DC lines were eliminated in a perforin-mediated CD8 Τ cell dependent and CD4 Τ cell independent response. After multiple injections, DC overexpressing CTLA4 or active TGFp could break the immune response to these inherent antigens and induced DC tumorogenesis in wild type mice. The tumor outgrowth in C57BL/6 mice was nicely observed by double-transduced DC lines to express both luciferase and active TGFp. actTGFp-DC tumor was shown to recruit myeloid-derived suppressor cells, induce CD4+ CD25+ Foxp3+ regulatory Τ cells and induce the expression of the inhibitory receptor PD-1 on tumor- infiltrating CD8+ Τ cells in order to escape tumor immunity. Tolerogenic DC lines were also tested for the induction of tolerance in a murine model of autoimmune disease, the experimental autoimmune encephalitis (EAE) model for human multiple sclerosis. EAE was induced in C57BL/6 mice by the adoptive transfer of lymph node cells isolated from donor mice previously immunized by a protein specific to the central nervous system, the myelin oligodendrocyte glycoprotein (MOG) emulsified in the complete freund adjuvant. The vaccination of donor and recipient mice with MOG-pulsed actTGFP-DC line prevented EAE induction. We are still investigating how the active TGFP protect mice from EAE development. We generated tolerogenic DC lines inducing tolerance in vitro and in vivo. Thereby these MuTu DC lines represent a great tool to explore the benefits of various immuno-modulatory molecules to manipulate the immune system toward a tolerogenic phenotype.

In vitro negative selection of viral superantigen-reactive thymocytes by thymic dendritic cells.

Intrathymic expression of endogenous mouse mammary tumor virus (MMTV)-encoded superantigens (SAg) induces the clonal deletion of T cells bearing SAg-reactive T-cell receptor (TCR) Vbeta elements. However, the identity of the thymic antigen-presenting cells (APC) involved in the induction of SAg tolerance remains to be defined. We have analyzed the potential of dendritic cells (DC) to mediate the clonal deletion of Mtv-7-reactive TCR alphabeta P14 transgenic thymocytes in an in vitro assay. Our results show that both thymic and splenic DC induced the deletion of TCR transgenic double positive (DP) thymocytes. DC appear to be more efficient than splenic B cells as negatively selecting APC in this experimental system. Interestingly, thymic and splenic DC display a differential ability to induce CD4+ SP thymocyte proliferation. These observations suggest that thymic DC may have an important role in the induction of SAg tolerance in vivo.

Phytogeographical origin of Madeiran common beans based on phaseolin patterns

The objective of this work was to determine the geographic origin of the Madeiran common bean (Phaseolus vulgaris) gene pool. Phaseolin patterns of 50 accessions representing the diversity of common bean collected in Madeira, Portugal, and conserved in the ISOPlexis Germplasm Bank, were analysed using the Experion automated electrophoresis system, based on lab-on-a-chip technology. Five common bean standard varieties with typical phaseolin patterns were used to determine the phytogeographical origin of the Madeiran common bean accessions. Ninety two percent of the accessions exhibited a phaseolin pattern consistent with the one of common bean types belonging to the Andean gene pool, while the origin of the remaining 8% of the accessions was indistinguishable. The application of a similarity coefficient of 85%, based on Pearson correlations, increases the number of accessions with uncertain pattern. The analytical approach used permitted the determination of the origin of the common bean gene pool, which is Andean in 98% of the cases, and clustering of the observed variability among the Madeiran common beans.

Face Short-Term Memory-Related Electroencephalographic Patterns can Differentiate Multi- versus Single-Domain Amnestic Mild Cognitive Impairment.

Amnestic mild cognitive impairment (aMCI) is characterized by memory deficits alone (single-domain, sd-aMCI) or associated with other cognitive disabilities (multi-domain, md-aMCI). The present study assessed the patterns of electroencephalographic (EEG) activity during the encoding and retrieval phases of short-term memory in these two aMCI subtypes, to identify potential functional differences according to the neuropsychological profile. Continuous EEG was recorded in 43 aMCI patients, whose 16 sd-aMCI and 27 md-aMCI, and 36 age-matched controls (EC) during delayed match-to-sample tasks for face and letter stimuli. At encoding, attended stimuli elicited parietal alpha (8-12 Hz) power decrease (desynchronization), whereas distracting stimuli were associated with alpha power increase (synchronization) over right central sites. No difference was observed in parietal alpha desynchronization among the three groups. For attended faces, the alpha synchronization underlying suppression of distracting letters was reduced in both aMCI subgroups, but more severely in md-aMCI cases that differed significantly from EC. At retrieval, the early N250r recognition effect was significantly reduced for faces in md-aMCI as compared to both sd-aMCI and EC. The results suggest a differential alteration of working memory cerebral processes for faces in the two aMCI subtypes, face covert recognition processes being specifically altered in md-aMCI.

Role of DC-SIGN in Lassa virus entry into human dendritic cells.

The arenavirus Lassa virus (LASV) causes a severe hemorrhagic fever with high mortality in humans. Antigen-presenting cells, in particular dendritic cells (DCs), are early and preferred targets of LASV, and their productive infection contributes to the virus-induced immunosuppression observed in fatal disease. Here, we characterized the role of the C-type lectin DC-specific ICAM-3-grabbing nonintegrin (DC-SIGN) in LASV entry into primary human DCs using a chimera of the prototypic arenavirus lymphocytic choriomeningitis virus (LCMV) expressing the LASV glycoprotein (rLCMV-LASVGP). We found that differentiation of human primary monocytes into DCs enhanced virus attachment and entry, concomitant with the upregulation of DC-SIGN. LASV and rLCMV-LASVGP bound to DC-SIGN via mannose sugars located on the N-terminal GP1 subunit of LASVGP. We provide evidence that DC-SIGN serves as an attachment factor for rLCMV-LASVGP in monocyte-derived immature dendritic cells (MDDC) and can accelerate the capture of free virus. However, in contrast to the phlebovirus Uukuniemi virus (UUKV), which uses DC-SIGN as an authentic entry receptor, productive infection with rLCMV-LASVGP was less dependent on DC-SIGN. In contrast to the DC-SIGN-mediated cell entry of UUKV, entry of rLCMV-LASVGP in MDDC was remarkably slow and depended on actin, indicating the use of different endocytotic pathways. In sum, our data reveal that DC-SIGN can facilitate cell entry of LASV in human MDDC but that its role seems distinct from the function as an authentic entry receptor reported for phleboviruses.

Psoriasis triggered by toll-like receptor 7 agonist imiquimod in the presence of dermal plasmacytoid dendritic cell precursors.

BACKGROUND: It has been proposed that the innate immune system plays a central role in driving the autoimmune T-cell cascade leading to psoriasis; however, there is no direct evidence for this. OBSERVATIONS: We observed aggravation and spreading of a psoriatic plaque when treated topically with the toll-like receptor (TLR) 7 agonist imiquimod. The exacerbation of psoriasis was accompanied by a massive induction of lesional type I interferon activity, detected by MxA expression after imiquimod therapy. Since imiquimod induces large amounts of type I interferon production from TLR7-expressing plasmacytoid dendritic cell precursors (PDCs), the natural interferon-producing cells of the peripheral blood, we asked whether PDCs are present in psoriatic skin. We identified high numbers of PDCs in psoriatic skin lesions (up to 16% of the total dermal infiltrate) based on their coexpression of BDCA2 and CD123. By contrast, PDCs were present at very low levels in atopic dermatitis and not detected in normal human skin. CONCLUSIONS: This study shows that psoriasis can be driven by the innate immune system through TLR ligation. Furthermore, our finding that large numbers of PDCs infiltrate psoriatic skin suggests a role of lesional PDCs as type I interferon-producing targets for the TLR7 agonist imiquimod.

Genetic diversity for gliadin patterns of durum wheat landraces in the Northwest of Iran and Azerbaijan

The objective of this study was to identify gliadin band patterns and the extent of genetic diversity in durum wheat genotypes from Northwestern Iran and the Republic of Azerbaijan. Gliadins from 46 landraces and four cultivars were evaluated through acid PAGE analyses. Sixty-six polymorphic bands and 81 patterns were identified. Twenty-four different motility bands and 22 patterns were found in the ω gliadin region with 14 polymorph bands and 20 patterns for α and γ gliadins, and 14 bands and 19 different patterns for β gliadins. The combination of these patterns generated 38 and 39 combinations for Gli-1 and Gli-2 loci, respectively. The genetic diversity index (H) was higher for α gliadins (0.924), followed by ω and γ gliadins (0.899 and 0.878, respectively), and for β gliadin patterns (0.866). Extensive polymorphism (H = 0.875) was observed in four gliadin pattern regions, with higher genetic diversity in the Iranian landraces than in the Azerbaijani ones. Each genotype had special identifying patterns in the gliadin acid PAGE analysis, and cluster analysis based on Jaccard's similarity coefficients formed six groups. Gliadin has a simple, repeatable and economic analysis, and can be used in genetic studies

Comment on 'The Molecular Evolutionary Patterns of the Insulin/FOXO Signaling Pathway'

Letter to the Editor on Wang M, Wang Q, Wang Z, Zhang X, Pan Y. The molecular evolutionary patterns of the insulin/FOXO signaling pathway

Recent patterns in gastric cancer: a global overview.

Until the mid-1990s, gastric cancer has been the first cause of cancer death worldwide, although rates had been declining for several decades and gastric cancer has become a relatively rare cancer in North America and in most Northern and Western Europe, but not in Eastern Europe, Russia and selected areas of Central and South America or East Asia. We analyzed gastric cancer mortality in Europe and other areas of the world from 1980 to 2005 using joinpoint regression analysis, and provided updated site-specific incidence rates from 51 selected registries. Over the last decade, the annual percent change (APC) in mortality rate was around -3, -4% for the major European countries. The APC were similar for the Republic of Korea (APC = -4.3%), Australia (-3.7%), the USA (-3.6%), Japan (-3.5%), Ukraine (-3%) and the Russian Federation (-2.8%). In Latin America, the decline was less marked, but constant with APC around -1.6% in Chile and Brazil, -2.3% in Argentina and Mexico and -2.6% in Colombia. Cancers in the fundus and pylorus are more common in high incidence and mortality areas and have been declining more than cardia gastric cancer. Steady downward trends persist in gastric cancer mortality worldwide even in middle aged population, and hence further appreciable declines are likely in the near future.

Current cardiovascular risk management patterns with special focus on lipid lowering in daily practice in Switzerland.

Background: There may be a considerable gap between LDL cholesterol (LDL-C) and blood pressure (BP) goal values recommended by the guidelines and results achieved in daily practice. Design Prospective cross-sectional survey of cardiovascular disease risk profiles and management with focus on lipid lowering and BP lowering in clinical practice. Methods: In phase 1, the cardiovascular risk of patients with known lipid profile visiting their general practitioner was anonymously assessed in accordance to the PROCAM-score. In phase 2, high-risk patients who did not achieve LDL-C goal less than 2.6 mmol/l in phase 1 could be further documented. Results: Six hundred thirty-five general practitioners collected the data of 23 892 patients with known lipid profile. Forty percent were high-risk patients (diabetes mellitus or coronary heart disease or PROCAM-score >20%), compared with 27% estimated by the physicians. Goal attainment rate was almost double for BP than for LDL-C in high-risk patients (62 vs. 37%). Both goals were attained by 25%. LDL-C values in phase 1 and 2 were available for 3097 high-risk patients not at LDL-C goal in phase 1; 32% of patients achieved LDL-C goal of less than 2.6 mmol/l after a mean of 17 weeks. The most successful strategies for LDL-C reduction were implemented in only 22% of the high-risk patients. Conclusion: Although patients at high cardiovascular risk were treated more intensively than low or medium risk patients, the majority remained insufficiently controlled, which is an incentive for intensified medical education. Adequate implementation of Swiss and International guidelines would expectedly contribute to improved achievement of LDL-C and BP goal values in daily practice.

Nutrient-based dietary patterns and the risk of head and neck cancer: a pooled analysis in the International Head and Neck Cancer Epidemiology consortium.

Background The association between dietary patterns and head and neck cancer has rarely been addressed. Patients and methods We used individual-level pooled data from five case-control studies (2452 cases and 5013 controls) participating in the International Head and Neck Cancer Epidemiology consortium. A posteriori dietary patterns were identified through a principal component factor analysis carried out on 24 nutrients derived from study-specific food-frequency questionnaires. Odds ratios (ORs) and corresponding 95% confidence intervals (CIs) were estimated using unconditional logistic regression models on quintiles of factor scores. Results We identified three major dietary patterns named 'animal products and cereals', 'antioxidant vitamins and fiber', and 'fats'. The 'antioxidant vitamins and fiber' pattern was inversely related to oral and pharyngeal cancer (OR = 0.57, 95% CI 0.43-0.76 for the highest versus the lowest score quintile). The 'animal products and cereals' pattern was positively associated with laryngeal cancer (OR = 1.54, 95% CI 1.12-2.11), whereas the 'fats' pattern was inversely associated with oral and pharyngeal cancer (OR = 0.78, 95% CI 0.63-0.97) and positively associated with laryngeal cancer (OR = 1.69, 95% CI 1.22-2.34). Conclusions These findings suggest that diets rich in animal products, cereals, and fats are positively related to laryngeal cancer, and those rich in fruit and vegetables inversely related to oral and pharyngeal cancer.