Thymic stromal lymphopoietin plays divergent roles in murine models of atopic and nonatopic airway inflammation.

BACKGROUND: Thymic stromal lymphopoietin (TSLP) is a cytokine primarily produced by epithelial cells, which has been shown to be a potent inducer of T-helper 2 (Th2)-type responses. However, TSLP has pleiotropic effects upon immune cells, and although extensively studied in the context of atopic asthma, its relevance as a therapeutic target and its role in the pathogenesis of nonatopic asthma remains unknown. We sought to investigate the role of TSLP in atopic, nonatopic and viral-induced exacerbations of pulmonary inflammation. METHODS: Using stringently defined murine models of atopic, nonatopic and virally exacerbated forms of pulmonary inflammation, we compared inflammatory responses of C57BL/6 wild-type (WT) and TSLP receptor-deficient (TSLPR KO) mice. RESULTS: Thymic stromal lymphopoietin receptor (TSLPR) signaling was crucial for the development of atopic asthma. Specifically, TSLPR signaling to lung recruited CD4+ T cells enhanced eosinophilia, goblet cell hyperplasia, and overall inflammation within the airways. In contrast, the absence of TSLPR signaling was associated with strikingly exaggerated pulmonary neutrophilic inflammation in a nonatopic model of airway inflammation. The inflammation was associated with excessive levels of interleukin (IL)-17A in the lungs, indicating that TSLP negatively regulates IL-17A. In addition, in a model of influenza-induced exacerbation of atopic airway inflammation, the absence of TSLPR signaling also led to exaggerated neutrophilic inflammation. CONCLUSION: Thymic stromal lymphopoietin plays divergent roles in the pathogenesis of atopic and nonatopic asthma phenotypes by either enhancing Th2 responses or curtailing T-helper 17 responses. These findings raise important caveats for the design of therapeutic interventions targeting TSLP in asthma.

IL-12-mediated STAT4 signaling and TCR signal strength cooperate in the induction of CD40L in human and mouse CD8(+) T cells.

CD40L is one of the key molecules bridging the activation of specific T cells and the maturation of professional and nonprofessional antigen-presenting cells including B cells. CD4(+) T cells have been regarded as the major T-cell subset that expresses CD40L upon cognate activation; however, we demonstrate here that a putative CD8(+) helper T-cell subset expressing CD40L is induced in human and murine CD8(+) T cells in vitro and in mice immunized with antigen-pulsed dendritic cells. IL-12 and STAT4-mediated signaling was the major instructive cytokine signal boosting the ability of CD8(+) T cells to express CD40L both in vitro and in vivo. Additionally, TCR signaling strength modulated CD40L expression in CD8(+) T cells after primary differentiation in vitro as well as in vivo. The induction of CD40L in CD8(+) T cells regulated by IL-12 and TCR signaling may enable CD8(+) T cells to respond autonomously of CD4(+) T cells. Thus, we propose that under proinflammatory conditions, a self-sustaining positive feedback loop could facilitate the efficient priming of T cells stimulated by high affinity peptide displaying APCs.

Genetic abnormalities and survival in multiple myeloma: the experience of the Intergroupe Francophone du Myélome.

Acquired genomic aberrations have been shown to significantly impact survival in several hematologic malignancies. We analyzed the prognostic value of the most frequent chromosomal changes in a large series of patients with newly diagnosed symptomatic myeloma prospectively enrolled in homogeneous therapeutic trials. All the 1064 patients enrolled in the IFM99 trials conducted by the Intergroupe Francophone du Myélome benefited from an interphase fluorescence in situ hybridization analysis performed on purified bone marrow plasma cells. They were systematically screened for the following genomic aberrations: del(13), t(11;14), t(4;14), hyperdiploidy, MYC translocations, and del(17p). Chromosomal changes were observed in 90% of the patients. The del(13), t(11;14), t(4;14), hyperdiploidy, MYC translocations, and del(17p) were present in 48%, 21%, 14%, 39%, 13%, and 11% of the patients, respectively. After a median follow-up of 41 months, univariate statistical analyses revealed that del(13), t(4;14), nonhyperdiploidy, and del(17p) negatively impacted both the event-free survival and the overall survival, whereas t(11;14) and MYC translocations did not influence the prognosis. Multivariate analyses on 513 patients annotated for all the parameters showed that only t(4;14) and del(17p) retained prognostic value for both the event-free and overall survivals. When compared with the currently used International Staging System, this prognostic model compares favorably. In myeloma, the genomic aberrations t(4;14) and del(17p), together with beta2-microglobulin level, are important independent predictors of survival. These findings have implications for the design of risk-adapted treatment strategies.

Transcriptional repression of peroxisome proliferator-activated receptor beta/delta in murine keratinocytes by CCAAT/enhancer-binding proteins.

The roles of peroxisome proliferator-activated receptors (PPARs) and CCAAT/enhancer-binding proteins (C/EBPs) in keratinocyte and sebocyte differentiation suggest that both families of transcription factors closely interact in the skin. Initial characterization of the mouse PPARbeta promoter revealed an AP-1 site that is crucial for the regulation of PPARbeta expression in response to inflammatory cytokines in the skin. We now present evidence for a novel regulatory mechanism of the expression of the PPARbeta gene by which two members of the C/EBP family of transcription factors inhibit its basal promoter activity in mouse keratinocytes. We first demonstrate that C/EBPalpha and C/EBPbeta, but not C/EBPdelta, inhibit the expression of PPARbeta through the recruitment of a transcriptional repressor complex containing HDAC-1 to a specific C/EBP binding site on the PPARbeta promoter. Consistent with this repression, the expression patterns of PPARbeta and C/EBPs are mutually exclusive in keratinocytes of the interfollicular epidermis and hair follicles in mouse developing skin. This work reveals the importance of the regulatory interplay between PPARbeta and C/EBP transcription factors in the control of proliferation and differentiation in this organ. Such insights are crucial for the understanding of the molecular control regulating the balance between proliferation and differentiation in many cell types including keratinocytes.

Inhibition and enhancement of in vitro helper activity of apocytochrome c-specific murine T cell populations and clones by anti-apocytochrome c-specific monoclonal antibodies.

A murine monoclonal antibody (mAb) specific for apocytochrome c was found to be able to either inhibit or enhance the helper activity of mouse apocytochrome c-specific T cell clones and populations in a hapten (trinitrophenyl)-carrier (apocytochrome c) system of T-B cell cooperation. This effect of the mAb was carrier specific, could not be ascribed simply to a shift in the kinetics of the antibody response and was observed using apocytochrome c T helper cells of different mouse haplotypes. In addition, the anti-apocytochrome c mAb was able to inhibit specific T helper cell activity even when the T cells were triggered with antigen-presenting cells pulsed with antigen. Taken together, these results suggested that the mAb was inhibiting helper activity due to its ability to modify the interaction between T cells and antigen-presenting cells.

The peroxisome proliferator-activated receptor (PPAR) β/δ agonist GW501516 inhibits IL-6-induced signal transducer and activator of transcription 3 (STAT3) activation and insulin resistance in human liver cells.

AIM/HYPOTHESIS: IL-6 induces insulin resistance by activating signal transducer and activator of transcription 3 (STAT3) and upregulating the transcription of its target gene SOCS3. Here we examined whether the peroxisome proliferator-activated receptor (PPAR)β/δ agonist GW501516 prevented activation of the IL-6-STAT3-suppressor of cytokine signalling 3 (SOCS3) pathway and insulin resistance in human hepatic HepG2 cells. METHODS: Studies were conducted with human HepG2 cells and livers from mice null for Pparβ/δ (also known as Ppard) and wild-type mice. RESULTS: GW501516 prevented IL-6-dependent reduction in insulin-stimulated v-akt murine thymoma viral oncogene homologue 1 (AKT) phosphorylation and in IRS-1 and IRS-2 protein levels. In addition, treatment with this drug abolished IL-6-induced STAT3 phosphorylation of Tyr⁷⁰⁵ and Ser⁷²⁷ and prevented the increase in SOCS3 caused by this cytokine. Moreover, GW501516 prevented IL-6-dependent induction of extracellular-related kinase 1/2 (ERK1/2), a serine-threonine protein kinase involved in serine STAT3 phosphorylation; the livers of Pparβ/δ-null mice showed increased Tyr⁷⁰⁵- and Ser⁷²⁷-STAT3 as well as phospho-ERK1/2 levels. Furthermore, drug treatment prevented the IL-6-dependent reduction in phosphorylated AMP-activated protein kinase (AMPK), a kinase reported to inhibit STAT3 phosphorylation on Tyr⁷⁰⁵. In agreement with the recovery in phospho-AMPK levels observed following GW501516 treatment, this drug increased the AMP/ATP ratio and decreased the ATP/ADP ratio. CONCLUSIONS/INTERPRETATION: Overall, our findings show that the PPARβ/δ activator GW501516 prevents IL-6-induced STAT3 activation by inhibiting ERK1/2 phosphorylation and preventing the reduction in phospho-AMPK levels. These effects of GW501516 may contribute to the prevention of cytokine-induced insulin resistance in hepatic cells.

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.

NLRC5 deficiency impairs MHC class I-dependent lymphocyte killing by cytotoxic T cells

Purpose/Objective: NLRs are intracellular proteins involved in sensing pathogen- and danger-associated molecular patterns, thereby initiating inflammatory responses or cell death. The function of the family member NLRC5 remains a matter of debate, particularly with respect to NF-jB activation, type I IFN, and MHC class I expression. Materials and methods: To study the function of this NLR in vivo, we generated Nlrc5-deficient mice. Results: We found that NLRC5 deletion led to a mild reduction in MHC class I expression on DCs and an intermediate decrease on B cells, while MHC class I levels were dramatically lowered on T, NKT, and NK cells. Nlrc5-/- lymphocytes showed decreased H-2 gene transcript abundance and, accordingly, NLRC5 was sufficient to drive MHC class I expression in a human lymphoid cell line. Moreover, endogenous NLRC5 localized to the nucleus and occupied the proximal promoter region of H-2 genes. Notably, cytotoxic T cell-mediated elimination of Nlrc5-/- lymphocytes was markedly reduced. In addition, we observed low NLRC5 expression in several murine and human lymphoid-derived tumor cell lines. Conclusions: We found that NLRC5 acts as a key transcriptional regulator of MHC class I genes, in particular in lymphocytes. Loss of NLRC5 expression represents an advantage for evading CD8+ T cellmediated elimination by downmodulation of MHCI levels * a mechanism transformed cells may take advantage of. Therefore, our data support an essential role for NLRs in directing not only innate, but also adaptive immune responses (Staehli F et al. J Immunol 2012).

CD4+CD25+ T cell depletion impairs tolerance induction in a murine model of asthma.

BACKGROUND: Regulatory T cells (Tregs) are key players in controlling the development of airway inflammation. However, their role in the mechanisms leading to tolerance in established allergic asthma is unclear. OBJECTIVE: To examine the role of Tregs in tolerance induction in a murine model of asthma. METHODS: Ovalbumin (OVA) sensitized asthmatic mice were depleted or not of CD25(+) T cells by anti-CD25 PC61 monoclonal antibody (mAb) before intranasal treatment (INT) with OVA, then challenged with OVA aerosol. To further evaluate the respective regulatory activity of CD4(+)CD25(+) and CD4(+)CD25(-) T cells, both T cell subsets were transferred from tolerized or non-tolerized animals to asthmatic recipients. Bronchoalveolar lavage fluid (BALF), T cell proliferation and cytokine secretion were examined. RESULTS: Intranasal treatment with OVA led to increased levels of IL-10, TGF-beta and IL-17 in lung homogenates, inhibition of eosinophil recruitment into the BALF and antigen specific T cell hyporesponsiveness. CD4(+)CD25(+)Foxp3(+) T cells were markedly upregulated in lungs and suppressed in vitro and in vivo OVA-specific T cell responses. Depletion of CD25(+) cells before OVA INT severely hampered tolerance induction as indicated by a strong recruitment of eosinophils into BALF and a vigorous T cell response to OVA upon challenge. However, the transfer of CD4(+)CD25(-) T cells not only suppressed antigen specific T cell responsiveness but also significantly reduced eosinophil recruitment as opposed to CD4(+)CD25(+) T cells. As compared with control mice, a significantly higher proportion of CD4(+)CD25(-) T cells from OVA treated mice expressed mTGF-beta. CONCLUSION: Both CD4(+)CD25(+) and CD4(+)CD25(-) T cells appear to be essential to tolerance induction. The relationship between both subsets and the mechanisms of their regulatory activity will have to be further analyzed.

Role of neutrophils in the early shaping of the Leishmania major specific immune response in experimental murine cutaneous Leishmaniasis

The development of a protective immune response to microorganisms involves complex interactions between the host and the pathogen. The murine model of infection with Leishmania major (L. major) allows the study of the factors leading to the development of a protective immune response. Following infection with the protozoan parasite L. major, most strains of mice heal their lesions, while a few fail to control infection, both processes linked to the development of specific T helper subsets. The early events occurring during the first days following parasite inoculation are thought to be critical in the development of the Leishmania-specific immune response. Neutrophils are the first cells arriving massively to the site of infection, and recent evidence points to their role as organizers of the immune response, yet their specific role in this process remains elusive. Through interactions with cells present at the parasite inoculation site, and possibly within the draining lymph nodes, neutrophils could have an impact not only on the recruitment of inflammatory cells but also on the activation of local as well as newly migrated cells that will be crucial in shaping the Leishmania-specific immune response.

Derivation of traceable and transplantable photoreceptors from mouse embryonic stem cells.

Retinal degenerative diseases resulting in the loss of photoreceptors are one of the major causes of blindness. Photoreceptor replacement therapy is a promising treatment because the transplantation of retina-derived photoreceptors can be applied now to different murine retinopathies to restore visual function. To have an unlimited source of photoreceptors, we derived a transgenic embryonic stem cell (ESC) line in which the Crx-GFP transgene is expressed in photoreceptors and assessed the capacity of a 3D culture protocol to produce integration-competent photoreceptors. This culture system allows the production of a large number of photoreceptors recapitulating the in vivo development. After transplantation, integrated cells showed the typical morphology of mature rods bearing external segments and ribbon synapses. We conclude that a 3D protocol coupled with ESCs provides a safe and renewable source of photoreceptors displaying a development and transplantation competence comparable to photoreceptors from age-matched retinas.

Between Immunology And Tolerance: Controlling Immune Responses Employing Tolerogenic Dendritic Cells

Dendritic cells (DCs) are the most efficient antigen presenting cells, they provide co-stimulation, are able to secrete various proinflammatory cytokines and therefore play a pivotal role in shaping adaptive immune responses. Moreover, they are important for the promotion and maintenance of central and peripheral tolerance through several mechanisms like the induction of anergy or apoptosis in effector T cells or by promoting regulatory T cells. The murine CD8α+ (MuTu) dendritic cell line was previously derived and described in our laboratory. The MuTu cell line has been shown to maintain phenotypical and functional characteristics of endogenous CD8α+ DCs. They are able to cross-present exogenous antigens to CD8+ T cells and produce interleukin (IL-) 12 upon engagement of Toll like receptors. The cell line constitutes an infinite source of homogenous, phenotypically well-defined dendritic cells. This allows us to investigate the role and potential of specific molecules in the induction as well as regulation of immune responses by DCs in a rational and standardized way. In a first project the MuTu dendritic cell line was transduced in order to stably express the immunosuppressive molecules IL-10, IL-35 or the active form of TGF-β (termed IL-10+DC, IL-35+DC or actTGFβ+DC). We investigated the capability of these potentially suppressive or tolerogenic dendritic cell lines to induce immune tolerance and explore the mechanisms behind tolerance induction. The expression of TGF-β by the DC line did not affect the phenotype of the DCs itself. In contrast, IL-10+ and IL-35+DCs were found to exhibit lower expression of co-stimulatory molecules and MHC class I and II, as well as reduced secretion of pro-inflammatory cytokines upon activation. In vitro co-culture with IL-35+, IL10+ or active TGFβ+ DCs interfered with function and proliferation of CD4+ and CD8+ T cells. Furthermore, IL-35 and active TGF-β expressing DC lines induced regulatory phenotype on CD4+ T cells in vitro without or with expression of Foxp3, respectively. In different murine cancer models, vaccination with IL-35 or active TGF-β expressing DCs resulted in faster tumor growth. Interestingly, accelerated tumor growth could be observed when IL-35-expressing DCs were injected into T cell-deficient RAG-/- mice. IL-10expressing DCs however, were found to rather delay tumor growth. Besides the mentioned autocrine effects of IL-35 expression on the DC line itself, we surprisingly observed that the expression of IL-35 or the addition of IL-35 containing medium enhances neutrophil survival and induces proliferation of endothelial cells. Our findings indicate that the cytokine IL-35 might not only be a potent regulator of adaptive immune responses, but it also implies IL-35 to mediate diverse effects on an array of cellular targets. This abilities make IL-35 a promising target molecule not only for the treatment of auto-inflammatory disease but also to improve anti-cancer immunotherapies. Indeed, by applying active TGFβ+ in murine autoimmune encephalitis we were able to completely inhibit the development of the disease, whereas IL-35+DCs reduced disease incidence and severity. Furthermore, the preventive transfer of IL-35+DCs delayed rejection of transplanted skin to the same extend as the combination of IL-10/actTGF-β expressing DCs. Thus, the expression of a single tolerogenic molecule can be sufficient to interfere with the adequate activation and function of dendritic cells and of co-cultured T lymphocytes. The respective mechanisms of tolerance induction seem to be different for each of the investigated molecule. The application of a combination of multiple tolerogenic molecules might therefore evoke synergistic effects in order to overcome (auto-) immunity. In a second project we tried to improve the immunogenicity of dendritic cell-based cancer vaccines using two different approaches. First, the C57BL/6 derived MuTu dendritic cell line was genetically modified in order to express the MHC class I molecule H-2Kd. We hypothesized that the expression of BALB/c specific MHC class I haplotype (H-2Kd) should allow the priming of tumor-specific CD8+ T cells by the otherwise allogeneic dendritic cells. At the same time, the transfer of these H-2Kd+ DCs into BALB/c mice was thought to evoke a strong inflammatory environment that might act as an "adjuvant", helping to overcome tumor induced immune suppression. Using this so called "semi-allogeneic" vaccination approach, we could demonstrate that the delivery of tumor lysate pulsed H-2Kd+ DCs significantly delayed tumor growth when compared to autologous or allogeneic vaccination. However, we were not able to coherently elucidate the cellular mechanisms underlying the observed effect. Second, we generated MuTu DC lines which stably express the pro-inflammatory cytokines IL-2, IL-12 or IL-15. We investigated whether the combination of DC vaccination and local delivery of pro-inflammatory cytokines might enhance tumor specific T cell responses. Indeed, we observed an enhanced T cell proliferation and activation when they were cocultured in vitro with IL-12 or IL-2-expressing DCs. But unfortunately we could not observe a beneficial or even synergistic impact on tumor development when cytokine delivery was combined with semi-allogeneic DC vaccination.

Fluence plays a critical role on the subsequent distribution of chemotherapy and tumor growth delay in murine mesothelioma xenografts pre-treated by photodynamic therapy.

BACKGROUND: The pre-conditioning of tumor vessels by low-dose photodynamic therapy (L-PDT) was shown to enhance the distribution of chemotherapy in different tumor types. However, how light dose affects drug distribution and tumor response is unknown. Here we determined the effect of L-PDT fluence on vascular transport in human mesothelioma xenografts. The best L-PDT conditions regarding drug transport were then combined with Lipoplatin(®) to determine tumor response. in vivo. Lasers Surg. Med. 47:323-330, 2015. © 2015 Wiley Periodicals, Inc. METHODS: Nude mice bearing dorsal skinfold chambers were implanted with H-Meso1 cells. Tumors were treated by Visudyne(®) -mediated photodynamic therapy with 100 mW/cm(2) fluence rate and a variable fluence (5, 10, 30, and 50 J/cm(2) ). FITC-Dextran (FITC-D) distribution was assessed in real time in tumor and normal tissues. Tumor response was then determined with best L-PDT conditions combined to Lipoplatin(®) and compared to controls in luciferase expressing H-Meso1 tumors by size and whole body bioluminescence assessment (n = 7/group). RESULTS: Tumor uptake of FITC-D following L-PDT was significantly enhanced by 10-fold in the 10 J/cm(2) but not in the 5, 30, and 50 J/cm(2) groups compared to controls. Normal surrounding tissue uptake of FITC-D following L-PDT was significantly enhanced in the 30 J/cm(2) and 50 J/cm(2) groups compared to controls. Altogether, the FITC-D tumor to normal tissue ratio was significantly higher in the 10 J/cm(2) group compared others. Tumor growth was significantly delayed in animals treated by 10 J/cm2-L-PDT combined to Lipoplatin(®) compared to controls. CONCLUSIONS: Fluence of L-PDT is critical for the optimal distribution and effect of subsequently administered chemotherapy. These findings have an importance for the clinical translation of the vascular L-PDT concept in the clinics. Lasers Surg. Med. 47:323-330, 2015.

Large T Antigen-Specific Cytotoxic T Cells Protect Against Dendritic Cell Tumors through Perforin-Mediated Mechanisms Independent of CD4 T Cell Help.

Our newly generated murine tumor dendritic cell (MuTuDC) lines, generated from tumors developing in transgenic mice expressing the simian virus 40 large T antigen (SV40LgT) and GFP under the DC specific promoter CD11c, reproduce the phenotypic and functional properties of splenic wild type CD8α(+) conventional DCs. They have an immature phenotype with low co-stimulation molecule expression (CD40, CD70, CD80, and CD86) that is upregulated after activation with toll-like receptor ligands. We observed that after transfer into syngeneic C57BL/6 mice, MuTuDC lines were quickly rejected. Tumors grew efficiently in large T transgene-tolerant mice. To investigate the immune response toward the large T antigen that leads to rejection of the MuTuDC lines, they were genetically engineered by lentiviral transduction to express luciferase and tested for the induction of DC tumors after adoptive transfer in various gene deficient recipient mice. Here, we document that the MuTuDC line was rejected in C57BL/6 mice by a CD4 T cell help-independent, perforin-mediated CD8 T cell response to the SV40LgT without pre-activation or co-injection of adjuvants. Using depleting anti-CD8β antibodies, we were able to induce efficient tumor growth in C57BL/6 mice. These results are important for researchers who want to use the MuTuDC lines for in vivo studies.