The dichotomy between type 1 and 2 conventional DCs under steady-state conditions is well defined. Bosteels et al. demonstrate that, upon inflammation, cDC2s acquire a hybrid inf-cDC2 phenotype, sharing phenotype, gene expression, and function with cDC1s and monocyte-derived cells, to optimally boost CD4 and CD8 immunity via Fc receptors.

No description available.

Bacterial biofilms represent a challenge to the healthcare system because of their resilience against antimicrobials and immune attack. Biofilms consist of bacterial aggregates embedded in an extracellular polymeric substance (EPS) composed of polysaccharides, nucleic acids and proteins. We hypothesised that carbohydrates could contribute to immune recognition of Pseudomonas aeruginosa biofilms by engaging C-type lectins. Here we show binding of Dendritic Cell-Specific Intercellular adhesion molecule-3-Grabbing Non-integrin (DC-SIGN, CD209), mannose receptor (MR, CD206) and Dectin-2 to P. aeruginosa biofilms. We also demonstrate that DC-SIGN, unlike MR and Dectin-2, recognises planktonic P. aeruginosa cultures and this interaction depends on the presence of the common polysaccharide antigen. Within biofilms DC-SIGN, Dectin-2 and MR ligands appear as discrete clusters with dispersed DC-SIGN ligands also found among bacterial aggregates. DC-SIGN, MR and Dectin-2 bind to carbohydrates purified from P. aeruginosa biofilms, particularly the high molecular weight fraction (HMW; >132,000 Da), with Ks in the nM range. These HMW carbohydrates contain 74.9-80.9% mannose, display α-mannan segments, interfere with the endocytic activity of cell-associated DC-SIGN and MR and inhibit Dectin-2-mediated cellular activation. In addition, biofilm carbohydrates reduce the association of the DC-SIGN ligand Lewis, but not fucose, to human monocyte-derived dendritic cells (moDCs), and alter moDC morphology without affecting early cytokine production in response to lipopolysaccharide or P. aeruginosa cultures. This work identifies the presence of ligands for three important C-type lectins within P. aeruginosa biofilm structures and purified biofilm carbohydrates and highlights the potential for these receptors to impact immunity to P. aeruginosa infection.

Lamellar cast iron is a very important technical alloy and the most used material in the casting production, and especially in the automotive industry which is the major consumer. Beside the many great properties, it is inclined to form casting defects of which some can be prevented, and some may be repaired subsequently. Shrinkage porosity is a randomly returning problem, which is difficult to understand and to avoid. This defect is a volumetric deficiency which appear as cavities inside the casting in connection to the casting surface. Another frequent defect is the metal expansion penetration. This defect is a material surplus squeezed to the casting surface containing sand inclusion from the mold material. Shrinkage porosity is usually mentioned together with metal expansion penetration as the formation mechanism of both defects have common roots. It is also generally agreed, that these type of defects are related to the volumetric changes occurring during solidification. Additionally, the formation of these defects are in connection with the coherency of the primary austenite dendrites. The purpose of this work was to develop knowledge on factors affecting a volume-change related casting defect formation in order to minimize the presence of these defects in engine component production. This was done by extending the existing solidification investigation methods with novel solutions. Introduction of expansion force measurement in the determination of dendrite coherency combined with multi axial volume change measurement refine the interpretation of the solidification. Comparison of registered axial and radial linear deformation in cylindrical samples indicated an anisotropic volume change. Different methods for dendrite coherency determination have been compared. It was shown that the coherency develops over an interval. Dependent on the added inoculant the coherency is reached at different levels of fractions of a solidified primary phase. It is also shown, that inoculation has an effect on the nucleation and growth of the primary phase. Quantitative image analysis has been performed on the primary phase in special designed samples designed to provoke shrinkage porosity and metal expansion penetration. It was found, that the inter-dendritic space varies within a casting. This was explained by the coarsening of the primary dendrites which originates from differences in the local time of solidification. / <p>QC 20131210</p>

Early in the course of human immunodeficiency virus (HIV) infection a population of latently infected cells is established which persists despite long-term anti-retroviral treatment. This latent reservoir of HIV-infected cells, which reflects mechanisms of transcriptional repression, is the major barrier to cure. Efforts to target the latent reservoir have been inefficient, indicating a need for a more complete understanding of how HIV transcription is regulated. The molecular networks involved in the regulation of HIV transcription remain incompletely defined. I hypothesized that utilization of a high throughput enhanced yeast one-hybrid assay would reveal novel host transcription factor-long terminal repeat (LTR) interactions and transcriptional networks that regulate HIV. The screen identified 42 human transcription factors and 85 total protein-DNA interactions with HIV LTRs. I investigated a subset of these factors for transcriptional activity in cell-based models of infection. Krüppel-like factors 2 and 3 (KLF2 and KLF3) are repressors of HIV-1 and HIV-2 transcription whereas PLAG1-like zinc finger 1 (PLAGL1) is an activator of HIV-2 transcription. These factors regulate HIV expression through direct protein-DNA interactions and correlate with epigenetic modifications of the HIV LTR. Multiple signals converging from the cellular environment and cell-cell interactions converge at the HIV LTR to determine HIV replication and transcription. Previous work in our lab has shown that strong signaling through the T cell receptor (TCR) was required to support HIV expression and the establishment of an inducible latent infection, whereas weak TCR signaling was insufficient for these outcomes. I hypothesized that dendritic cells-CD4+ T cell interactions provide signals that compensate for weak TCR signaling, supporting HIV-1 expression and generation of inducible latent infection. I used CD4+ T cells that express chimeric antigen receptors in a dendritic cell coculture model to deliver differential signals to CD4+ T cells during cell-to-cell transmission of HIV. I found that signals from dendritic cells compensate for weak TCR signaling, facilitating cell activation, HIV expression and establishment of an inducible infection.

Despite the lack of robust clinical response, treatment strategies for cancer-induced depression (CID) are currently limited to those developed for non-cancer-related depression. The work presented in this dissertation conceptualizes CID as a pathophysiologically distinct form of depression. To investigate CID at the most basic level, we first developed a preclinical model that was validated by comparison to an established model of stress-induced depressive-like behaviours. The positive control model was developed by chronically treating female BALB/c mice with oral corticosterone (CORT). The CID model was developed using subcutaneous inoculation with 4T1 mammary carcinoma cells. Anhedonia, behavioural despair, and dendritic atrophy in the medial prefrontal cortex (mPFC) were observed in both models. Similar to many human cancer cell lines, 4T1 cells were shown to secrete significant amounts of glutamate, which was markedly attenuated using the system xc- inhibitor sulfasalazine (SSZ). In CID mice, oral treatment with SSZ was at least as effective as fluoxetine, a popular clinical antidepressant, at preventing depressive-like behaviours. This effect was primarily attributable to intact SSZ, rather than its anti-inflammatory metabolite. RNA-sequencing was performed on hippocampal samples from CID and CORT animals. Analysis of differential expressed genes (DEGs) revealed significant overlap between the two models. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways and biological process gene ontologies (GO:BP) terms related to ion homeostasis and neuronal communication were enriched for both models. CID was associated with additional DEGs that were not identified in the CORT model. These DEGs were enriched in KEGG pathways and GO:BP terms related to neuronal development, intracellular signalling cascade, learning, and memory. These studies suggest that CID may involve a distinct aetiology, and that glutamate secretion by cancer cells presents a viable target for antidepressant treatment. The development of mechanism-based therapeutics for CID will dramatically improve the quality of life for cancer patients. / Thesis / Doctor of Philosophy (PhD) / Cancer patients are at a high risk of developing depression. In addition to the psychological stress caused by a cancer diagnosis, there is evidence that cancer causes depression through biological pathways. To investigate these pathways, a mouse model of cancer-induced depression (CID) was developed. This model showed comparable behavioural and structural brain deficits to those observed in a stress model of depression. Cancer cells secrete elevated levels of glutamate, a signalling molecule that is involved in depression. In CID mice, inhibiting glutamate release had an antidepressant effect similar to that of fluoxetine, a standard clinical antidepressant. A genetic analysis on brain samples from the CID model revealed significant overlap with the stress model of depression. CID mice had additional changes relevant to learning, memory, and brain cell development that were not detected in the stress model. A better understanding of CID will lead to better treatment strategies developed specifically for cancer patients.

Background: Patients treated with an inhibitor of the mechanistic target of rapamycin (mTORI) in a calcineurin inhibitor (CNI)-free immunosuppressive regimen after heart transplantation (HTx) show a higher risk for transplant rejection. We developed an immunological monitoring tool that may improve the identification of mTORI-treated patients at risk for rejection. Methods: Circulating dendritic cells (DCs) and regulatory T cells (Tregs) were analysed in 19 mTORI- and 20 CNI-treated HTx patients by flow cytometry. Principal component and cluster analysis were used to identify patients at risk for transplant rejection. Results: The percentages of total Tregs (p = 0.02) and CD39+ Tregs (p = 0.05) were higher in mTORI-treated patients than in CNI-treated patients. The principal component analysis revealed that BDCA1+, BDCA2+ and BDCA4+ DCs as well as total Tregs could distinguish between non-rejecting and rejecting mTORI-treated patients. Most mTORI-treated rejectors showed higher levels of BDCA2+ and BDCA4+ plasmacytoid DCs and lower levels of BDCA1+ myeloid DCs and Tregs than mTORI non-rejectors. Conclusion: An mTORI-based immunosuppressive regimen induced a sufficient, tolerance-promoting reaction in Tregs, but an insufficient, adverse effect in DCs. On the basis of patient-specific immunological profiles, we established a flow cytometry-based monitoring tool that may be helpful in identifying patients at risk for rejection.

La thérapie par cellules T à récepteur d'antigène chimérique (CAR) a produit d'incroyables réponses cliniques contre plusieurs tumeurs malignes, mais elle laisse une marge de manœuvre pour l'échappement de l'antigène. Une nouvelle approche pour surmonter ce problème consisterait à combiner la capacité des CAR à cibler les tumeurs avec la capacité des cellules dendritiques (CD) à amorcer les cellules T afin de générer une thérapie cellulaire qui favorise la propagation de l'épitope plutôt que la destruction directe de la cible. J'ai donc émis l'hypothèse que les cellules dendritiques exprimant les CAR (CAR-CD) peuvent renforcer l'amorçage des cellules T contre les cibles tumorales afin de produire un contrôle adaptatif des tumeurs médié par les cellules T. En utilisant des CD dérivées de la moelle osseuse murine, j'ai d'abord développé un nouveau protocole pour générer des CD. En ajoutant de l'IFNα aux cultures de DC GM-CSF, j'ai généré des CD qui expriment des niveaux plus élevés de molécules stimulant les cellules T et qui induisent une plus forte prolifération des cellules T CD8+ in-vitro par rapport aux CD générées avec le GM-CSF seul. In vivo, ces CD favorisent des réponses effectrices plus fortes, les cellules T CD8+ résultantes exprimant des niveaux plus élevés de marqueurs effectrices, notamment KLRG1 et TIM3, mais des niveaux plus faibles de molécules inhibitrices, notamment PD-1 et CD73. L'expression d'un CAR dans ces DC leur permet de tuer directement des cibles tumorales et d'acquérir des antigènes tumoraux. Plus important encore, ces CAR-CD sont en synergie avec les cellules T CD8+ pour contrôler les cellules tumorales in-vitro, en améliorant la prolifération et la capacité de destruction des cellules T. Chez la souris, les CAR-CD agissent comme un vaccin in vivo en favorisant la génération de réponses de cellules T spécifiques de la tumeur lorsqu'elles sont injectées par voie intratumorale, ce qui permet d'améliorer le contrôle de la tumeur. / Chimeric Antigen Receptor (CAR) T cell therapy has produced unbelievable clinical responses against several malignancies however, this therapy leaves room for antigen escape. One novel approach to overcome this would be to combine the tumor targeting ability of CARs with the T cell priming capacity of dendritic cells (DCs) to generate a cell therapy that provokes endogenous adaptive immunity through epitope spreading rather than just direct target killing. Therefore, I hypothesized that CAR expressing DCs (CAR-DCs) can enhance T cell priming against tumor targets to produce adaptive T cell mediated tumor control. Using murine bone marrow derived DCs, I first developed a new protocol for generating DCs using IFNα. The addition of IFNα to GM-CSF DC cultures generated DCs that express higher levels of T cell stimulatory molecules and induce stronger CD8+ T cell proliferation in-vitro compared to DCs generated with GM-CSF alone. In-vivo, these DCs promote stronger effector responses with the resulting CD8+ T cells expressing higher levels of effector markers including KLRG1 and TIM3 but lower levels of inhibitory molecules including PD-1 and CD73. The expression of a CAR in these DCs allowed them to directly kill tumor targets and acquire tumor antigens. More importantly, these CAR-DCs synergized with CD8+ T cells to control tumor cells in-vitro, enhancing the proliferation and killing capacity of the T cells. In mice, CAR-DCs act as an in-vivo vaccine promoting the generation of tumor specific T cell responses when injected intratumorally, producing enhanced tumor control.

Background: Extracorporeal photopheresis (ECP) induces immunological changes that lead to a reduced risk of transplant rejection. The aim of the present study was to determine optimum conditions for ECP treatment by analyzing a variety of toleranceinducing immune cells to optimize the treatment. Methods: Ten ECP treatments were applied to each of 17 heart-transplant patients from month 3 to month 9 post-HTx. Blood samples were taken at baseline, three times during treatment, and four months after the last ECP treatment. The abundance of subsets of tolerance-inducing regulatory T cells (Tregs) and dendritic cells (DCs) in the samples was determined by flow cytometry. A multivariate statistical model describing the immunological status of rejection-free heart transplanted patients was used to visualize the patient-specific immunological improvement induced by ECP. Results: All BDCA+ DC subsets (BDCA1+ DCs: p < 0.01, BDCA2+ DCs: p < 0.01, BDCA3+ DCs: p < 0.01, BDCA4+ DCs: p < 0.01) as well as total Tregs (p < 0.01) and CD39+ Tregs (p < 0.01) increased during ECP treatment, while CD62L+ Tregs decreased (p < 0.01). The cell surface expression level of BDCA1 (p < 0.01) and BDCA4 (p < 0.01) on DCs as well as of CD120b (p < 0.01) on Tregs increased during the study period, while CD62L expression on Tregs decreased significantly (p = 0.04). The cell surface expression level of BDCA2 (p = 0.47) and BDCA3 (p = 0.22) on DCs as well as of CD39 (p = 0.14) and CD147 (p = 0.08) on Tregs remained constant during the study period. A cluster analysis showed that ECP treatment led to a sustained immunological improvement. Conclusions: We developed an immune monitoring assay for ECP treatment after heart transplantation by analyzing changes in tolerance-inducing immune cells. This assay allowed differentiation of patients who did and did not show immunological improvement. Based on these results, we propose classification criteria that may allow optimization of the duration of ECP treatment.

No description available.