Un inconvénient majeur de l’utilisation des protéines thérapeutiques est leur immunogénicité,c'est-à-dire le déclenchement chez les patients d’une réponse immunitaire, avec production d’anticorps (anti-drug antibodies, ADA). Parmi les facteurs contributifs, les agrégats de protéines dans les spécialités administrées pourraient jouer un rôle majeur dans l’immunogénicité. Par ailleurs, la présence d’ADA de haute affinité et de divers isotypes suggère la mise en place d’une réponse immunitaire classique, faisant intervenir les cellules présentatrices d’antigènes et plus particulièrement les cellules dendritiques.Nous avons développé un modèle d’étude de l’impact d’agrégats de protéines sur la maturation de cellules dendritiques, dérivées de monocytes isolés du sang(moDC). Dans ce but, des agrégats d’hormone de croissance (GH) et d’anticorps (Rituximab) ou d’IgG1 polyclonale ont été produits et caractérisés.Nous avons montré que ces agrégats induisent la maturation des moDC, objectivée par une augmentation de l’expression de marqueurs d’activation et de co-stimulation (CD40, CD80,CD83, CD86 et HLA-DR), et par l’augmentation dela production de cytokines et chimiokines proinflammatoires (IL-6, IL-8,IL-12p40, CCL2, CCL3,CCL4 et CXCL10).En utilisant un modèle de co-culture allogénique,nous avons montré que les moDC stimulées par les agrégats induisent la prolifération de lymphocytes TCD4+, dont la polarisation dépendait de la nature de la protéine. Ainsi les agrégats de GH conduisent à une production majoritaire d’IFNγ, signe d’une réponse de type Th1, tandis que les agrégats d’anticorps induisent une réponse mixte, Th1, Th2,Th17 (production d’IFNγ, IL-5, IL-13 et IL-17.Enfin, nous avons commencé l’étude des mécanismes intra cellulaires impliqués dans l’activation des moDC, en montrant que les agrégats de GH induisent la phosphorylation de p38MAPK, ERK, JNK et NF-κB (p65). Ces mêmes voies de signalisation sont impliquées dans l’expression de CXCL10,chimiokine connue pour induire la polarisation Th1.Au final, ces résultats confirment l’effet immunomodulateur des agrégats de protéines sur les cellules dendritiques et précisent leur rôle de signal de danger conduisant à la mise en place d’une réponse immunitaire contre les protéines thérapeutiques. / A major drawback in therapeutic biological products (BP) use is the development of anti-drug antibodies (ADA) in patients. Among other factors, BP aggregates seems to play a major role in immunogenicity. Moreover, the presence of ADA with high affinity and different isotypes suggest a CD4 T-cell dependent immune response and therefore a pivotal role for antigen presenting cells, such as dendritic dells (DC).In order to determine if BP aggregates participate to DC activation, aggregates form human growth hormone (GH) and antibodies (Rituximab and polyclonal IgG1) were produced and characterized.Their impact was tested on a model of monocytederived dendritic cells (mo-DC).We have shown aggregates were able to induce moDC maturation, as observed with increase of key co-stimulatory and maturation markers (CD40, CD80, CD83, CD86 and HLA-DR), and by increase of pro-inflammatory cytokines and chemokines (IL-6, IL-8, IL-12p40, CCL2, CCL3, CCL4, CXCL10).Using an allogenic model of co-culture, we have shown that moDC stimulated with aggregates were able to induce CD4+ T cells proliferation.Polarization was different following the nature of the protein. GH aggregates were able to induceIFNγ, sign of Th1 response, whereas antibody aggregates induced Th1, Th2, Th17 mix response (with production of IFNγ, IL-5, IL-13 and IL-17).Finally, we started to study intracellular mechanisms involved in moDC activation, by showing that GH aggregates were able to induce p38MAPK, ERK, JNK and NF-κB (p65)phosphorylation. These pathways are involved in CXCL10 expression, which is implicated in Th1 polarization. These results confirmed the immunomodulary effect of protein aggregates on DC and their role as danger signal, inducing an immune response against therapeutic proteins.

Dendritic spines are the major sites for receiving excitatory synaptic inputs and play important roles in neuronal signal transduction, memory storage and neuronal circuit organization. Structural plasticity of dendritic spines is correlated with functional plasticity, and is critical for learning and memory. Visualization of the changes of dendritic spines at the ultrastructural level that specifically correlated with their function changes in high throughput would shed light on detailed mechanisms of synaptic plasticity. Here we developed a correlative light and electron microscopy workflow which combines two-photon MNI-glutamate uncaging, pre-embedding immunolabeling, Automatic Tape-collecting Ultramicrotome sectioning and scanning electron microscopy imaging. This method bridges two different visualization platforms, directly linking ultrastructure and function at the level of individual synapses. With this method, we successfully relocated single dendritic spines that underwent long-term potentiation (LTP) induced by two-photon MNI-glutamate uncaging, and visualized their ultrastructures and AMPA receptors distribution at different phases of LTP in high throughput. / Includes bibliography. / Dissertation (Ph.D.)--Florida Atlantic University, 2020. / FAU Electronic Theses and Dissertations Collection

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京都大学 / 0048 / 新制・課程博士 / 博士(医科学) / 甲第22122号 / 医科博第107号 / 新制||医科||7(附属図書館) / 京都大学大学院医学研究科医科学専攻 / (主査)教授 生田 宏一, 教授 濵﨑 洋子, 教授 杉田 昌彦 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22830号 / 医博第4669号 / 新制||医||1047(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 江藤 浩之, 教授 武藤 学, 教授 伊藤 貴浩 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Crosspresentation allows antigen presenting cells to present peptides from exogenously derived antigens onto MHC Class I for presentation to CD8+ T cells. Though this pathway shares key players with the Classical Class I and Class II pathways, several questions remain. A genomewide siRNA screen was performed to look for genes that selectively affected the crosspresentation or the Class II pathways. Among the genes identified in the screen was the Rab GTPase Rab39a. Rab39a was required for efficient crosspresentation but was dispensable for the presentation of endogenously expressed antigen. Both TAP-dependent and independent antigen required Rab39a for efficient presentation. Rab39a localized to late endosomes and phagosomes, though interestingly it was not required for the Class II pathway. Analysis of phagosomes from Rab39a KO or rescued cells has shown that in the presence of Rab39a, phagosomes were enriched for the open form of MHC Class I as well as TAP1, a member of the peptide loading complex. The enriched open form of MHC-I was peptide receptive, suggesting that it could contribute to crosspresentation. Phagosomes from Rab39a positive cells had reduced degradative capability and had increased levels of Sec22b, a SNARE protein reported to deliver ER-golgi sourced cargo to phagosomes. Furthermore, inhibition of ER-golgi transport via brefeldin A abolished the phenotype conferred by Rab39a. Thus, we hypothesize that Rab39a mediates the delivery of ER-golgi derived cargo to the antigen containing phagosome. This delivery allows peptide receptive MHC-I, as well as the peptide loading complex to reach the antigen, thereby facilitating crosspresentation.

Cell therapies are an emerging form of therapy, with the potential to treat and cure a variety of diseases. As more cell therapies become approved and commercialized, challenges remain in the manufacturing of these often single-batch products due to their complexity and patient-to-patient variability, which limit their cost-effectiveness and reproducibility. In this dissertation, we aim to improve the manufacturing of two different cell therapies, namely, organoid-based cell therapies using hydrogel scaffolds, and adoptive cell therapies using deep learning and microfluidics, to facilitate their widespread clinical use. First, we develop new tools to manufacture organoids, which are widespread in drug-screening technologies, but have been sparingly used for cell therapy as current approaches for producing self-organized cell clusters lack scalability or reproducibility. Here, we use alginate microwell scaffolds to form pre-vascularized organoids composed of endothelial cells and mesenchymal stem cells, where the size and structure can be readily tuned by varying the cell source, ratio of cells, or size of the microwells. Furthermore, by uncrosslinking the alginate scaffold, the organoids can be harvested in a gentle manner without damaging their structure or impairing their functionality. Finally, we assess the ability of the pre-vascularized organoids to restore vascular perfusion in a mouse model of hindlimb ischemia. By making use of the dynamic nature of hydrogels, this method can offer high yields of reproducible, self-organized multicellular aggregates for use in cell therapies. Next, we shift our focus to the identification of antigen-specific T cells, which is a critical step in the manufacturing of adoptive cell therapy. Conventional techniques for selecting antigen-specific T cells are time-consuming, making them difficult to adapt for large-scale manufacturing, and are limited to pre-defined antigenic peptide sequences. Here we train a deep learning model to rapidly classify videos of antigen-specific CD8+ T cells by distinguishing the distinct interaction dynamics (in motility and morphology) between cognate and non-cognate T cells and dendritic cells (DCs). The model is able to classify high affinity antigen-specific CD8+ T cells from OT-I mice with an area under the curve (AUC) of 0.91, and generalizes well to other types of high and low affinity CD8+ T cells. We also show that the experimental addition of anti-CD40 antibodies amplifies the differences between cognate and non-cognate T cells and DCs, thereby improving the model’s ability to discriminate between them. This workflow can be used to better understand the role of cognate T cell – DC interactions in the pathogenesis of cancer and autoimmune diseases, and can be integrated into a device to simplify and accelerate the selection of antigen-specific T cells for use in adoptive cell therapy. Finally, we sought to develop a device to address two other issues associated with the selection of antigen-specific T cells: low-throughput screening, and the inability to assess a mixed population of T cells against a library of antigens, both of which are necessary to identify rare T cells, and improve clinical outcomes of the corresponding cell therapy. A few specialized assays exist that can assess T cells against multiple antigens, but they are often limited by an increased manufacturing burden. Here, we develop a microfluidic artificial lymph node, which is inspired by the efficient selection of antigen-specific T cells in vivo. In particular, our flow-through design consists of multiple compartments, each containing microcarrier beads coated with DCs presenting a distinct antigen, such that T cells that are flowed sequentially through each compartment can stably arrest to cognate DCs, becoming captured in the appropriate compartment. We test a single-compartment device computationally using agent-based simulations, and experimentally using a mixed population of antigen-specific and wild-type (WT) (non-specific) T cells, and in both cases we observe a preferential accumulation of cognate, antigen-specific T cells. This proof-of-concept single-compartment device can be readily scaled up to systematically test many T cells against multiple antigens. Underlying this work is the development of technologies to enable the large-scale manufacturing of cell therapies. Cell therapies are undergoing a transformation to a new class of therapeutic modality, and there are many emerging questions, especially related to the scale-up and scale-out of production processes. Together, this work aims to engineer technologies to improve cell therapy manufacturing processes, facilitate their clinical translation, and ensure their availability to all patients who would benefit from them.

Content: Environmental pollution caused by leather making is the primary concern in the development of leather industry. The use of safe, effective and multi-functional green chemical products has the advantages of reducing leather operations, increasing chemicals utilization, decreasing the environmental burden, improving leather quality. In this study, dendritic-linear polymers of PAMAM-Si 1G and PAMAM-Si 2G were applied to fatliquoring process, which were prepared by branching polysiloxane on the dendritic polyamide-amine (PAMAM). Then the emulsion properties, fatliquoring properties and fatliquoring mechanism were studied by EDS, SEM, XRD, TG and washing experiments. The conclusion was drawn that PAMAM-Si are weak alkali products with high emulsion stability. The particle size of PAMAM-Si 1G was 35.8 nm, and that of PAMAM-Si 2G was 26.7 nm. They can improve the softness, shrinkage temperature and physical and mechanical properties of leather. The softness of leather with PAMAM-Si 1G and PAMAM-Si 2G increased by 115.6% and 104.7% respectively. The shrinkage temperature of leather with PAMAM-Si 2G increased by 2.9℃. The Breaking elongation of leather with PAMAM-Si 1G and PAMAM-Si 2G increased by 38.6% and 32.4% respectively. At the same time, PAMAM-Si not only increased the distance and disorder of fiber but combined with collagen fiber through hydrogen bond, a certain amount of physical adsorption and covalent bond. Take-Away: 1. The dendritic-linear polymers of PAMAM-Si 1G and PAMAM-Si 2G were prepared by branching polysiloxane on the dendritic polyamide-amine (PAMAM). 2. PAMAM-Si can improve the softness, shrinkage temperature and physical and mechanical properties of leather. 3. PAMAM-Si not only increased the distance and disorder of fiber but combined with collagen fiber through hydrogen bond, a certain amount of physical adsorption and covalent bond.

The diploma thesis is focused on the study of the influence of selected organic additives on the properties and morphology of zinc deposit on the negative electrode when used in zinc-air accumulators. Organic additives have been selected based on the study of literature and previously done experiments. The deposition was made on the tin plate electrodes in a solution of 6 mol/l KOH saturated with ZnO with added additives. All chosen additives were studied with a focus on the morphology of zinc deposit and with regard to their ability to suppress dendritic growth. Zinc morphology was studied by using a Tescan Vega 3 electron microscope and a Rigaku MiniFlex HR 600 X-ray diffractometer.

L’immunogénicité des biothérapies constitue une limitation majeure au traitement des patients atteints de maladies chroniques et se traduit par la production d’anticorps dirigés contre le biomédicament (anti-drug antibodies, ADA). La détection d’ADA de haute affinité et d’isotypes divers chez les patients suggère la mise en place d’une réponse immunitaire adaptative classique orchestrée par les cellules dendritiques. Par ailleurs, la présence d’agrégats protéiques dans les spécialités administrées serait un des facteurs favorisant l’immunogénicité, ces agrégats pouvant jouer le rôle de signal de danger.L’objectif de notre travail était de mieux comprendre les interactions des agrégats de protéines avec les cellules dendritiques et les lymphocytes T aboutissant au déclenchement d’une réponse immunitaire adaptative spécifique nécessaire à la production d’ADA.Dans un premier temps, nous avons montré que des agrégats d’infliximab, anticorps monoclonal anti-TNFa;, induisaient la maturation de cellules dendritiques humaines dérivées de monocytes (moDC). Celle-ci se traduit par l’augmentation de l’expression de marqueurs membranaires d’activation et de costimulation et la sécrétion de cytokines et chimiokines pro-inflammatoires. Nous avons également montré que ces modifications phénotypiques induites par les agrégats favorisaient la prolifération de lymphocytes T CD4+ et la production de cytokines. Par la suite, nous avons décrit les mécanismes cellulaires précoces impliqués dans l’activation de ces cellules en montrant que la neutralisation du récepteur FcgRIIa et de la tyrosine kinase Syk inhibait la maturation des moDC ainsi que l’activation des lymphocytes T CD4+.Par ailleurs, nous avons évalué le rôle des agrégats d’infliximab dans la génération de néo-épitopes, en mettant en évidence l’existence d’un répertoire de lymphocytes T CD4+ naïfs reconnaissant spécifiquement les agrégats d’infliximab, chez le sujet sain. Ainsi, grâce à des préparations d’agrégats bien caractérisées et d’un modèle de co-cultures autologues de lymphocytes T CD4+ naïfs et de moDC chargées avec les agrégats, nous avons montré que la fréquence de LT CD4+ naïfs spécifiques des agrégats d’infliximab est plus importante que celle retrouvée pour l’anticorps natif. Ces résultats suggèrent une présentation accrue d'épitopes et de néo-épitopes dérivant des agrégats d'infliximab.In fine, ce travail contribue à une meilleure compréhension des conséquences biologiques de l’agrégation des protéines à visée thérapeutique sur le déclenchement de la réponse immunitaire adaptative spécifique en mettant l’accent sur les rôles adjuvant et antigénique des agrégats protéiques. / Immunogenicity of biotherapeutic proteins is a major drawback in the treatment of patients with chronic diseases characterized by the production of anti-drug antibodies (ADA). The detection of ADA with high affinity and of various isotypes suggests a CD4 T cell-dependent adaptive immune response with a pivotal role for dendritic cells. Among other factors, the presence of protein aggregates in the administered products can promote immunogenicity, as aggregates seem to act as danger signals.The aim of our work is to better understand the interactions of protein aggregates with dendritic cells and T cells, leading to the establishment of a specific adaptive immune response needed for the production of ADA.We first showed that aggregation of infliximab, a monoclonal anti-TNFa; antibody, induced the maturation of human monocyte-derived dendritic cells (moDC) via an increase in the expression of activation and costimulatory surface markers and the secretion of pro-inflammatory cytokines and chemokines. Moreover, we showed that these phenotypic changes induced by aggregates promote CD4 T-cell proliferation and cytokine production. Subsequently, we described the early events involved in moDC and T-cell response by showing that the neutralization of the FcgRIIa receptor and the tyrosine kinase Syk inhibited moDC maturation and CD4 T-cell activation.Furthermore, we evaluated the involvement of infliximab aggregates in the generation of neo-epitopes by identifying a naïve CD4 T-cell repertoire recognizing infliximab aggregates in healthy subjects. By testing well characterized aggregate preparations and using an autologous co-culture model of naïve CD4 T cells and moDC loaded with aggregates, we showed that the frequency of naïve CD4 T cells specific for infliximab aggregates was higher than the one found for the native antibody. These results suggested an increased presentation of epitopes and neo-epitopes derived from infliximab aggregates.In resume, our work contributes to a better understanding of the biological consequences of therapeutic protein aggregation on the onset of the specific adaptive immune response by focusing on the adjuvant and antigenic role of protein aggregates.