2 research outputs found

    Adjuvant-loaded protein-based nanocapsules induce effective anti-cancer immunity

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    Nanocarrier-based vaccines enable the simultaneous transport of antigens and adjuvants for specific activation of the immune system for cancer therapy. In this context, the targeting of dendritic cells (DCs), whose maturation can be induced and directed by specific adjuvants and which can prime naϊve T cells in an antigen-specific manner, is particularly relevant. Within the scope of this PhD project, protein-based nanocapsules with a shell/core morphology were evaluated. Human serum albumin (HSA) and ovalbumin (OVA) with high biocompatibility, degradability, and low cytotoxicity were used as shell materials. Combinatorial encapsulation of multiple adjuvants, which stimulate different signaling pathways, into the aqueous capsule core was successfully established. The uptake of HSA capsules loaded with the adjuvants muramyl dipeptide (MDP), resiquimod (R848) and polyinosinic:polycytidylic acid (Poly(I:C)) by dendritic cells and the subsequent DC maturation were demonstrated. Additionally, it could be shown that the combination of adjuvants in particular is necessary to sufficiently stimulate DCs. Flow cytometric analyses were performed to evaluate the expression of costimulatory molecules and the secretion of pro-inflammatory cytokines and chemokines. OVA-NCs allowed the simultaneous transport of the model antigen with the adjuvants MDP, R848, and Poly(I:C) to dendritic cells. Effective OVA peptide presentation to naïve T cells via loading on MHC class I and MHC class II molecules inducing T cell proliferation could be demonstrated in vitro. Subsequently, the potential of adjuvant-loaded OVA nanocapsules to induce anti-tumor immune responses was evaluated in a murine melanoma model. For this purpose, C57BL/6J mice were injected with OVA-expressing B16/F10 melanoma cells and subsequently treated with different OVA-NC formulations. Significant reduction of tumor growth, in particular by combined encapsulating of R848 and MDP, was obtained. Furthermore, it could be shown that only the transport of antigen and adjuvants in nanocapsules to DCs induced an efficient anti-tumor immune response, whereas the administration of soluble adjuvants and antigens did not achieve comparable results. Through a comparative study including different NC amounts and injection routes, the optimal therapeutic regimen was established. To induce complete tumor remission, R848, a Toll-like receptor 7/8 agonist, was encapsulated in combination with the potent STING agonist diamidobenzimidazole (diABZI, compound 3), in OVA nanocapsules. This adjuvant combination induced synergistic effects in vitro with respect to the expression of DC maturation markers as well as the production of a broad spectrum of pro-inflammatory cytokines and chemokines. In particular, the induction of type I interferons, which are required for an efficient anti-tumor immune response, by diABZI offered an advantage over the previously established adjuvant combination. In subsequent tumor studies, animals with OVA-expressing B16/F10 melanomas were cured by triple injection of diABZI- and R848/diABZI-loaded nanocapsules. The nanovaccine was evolved by supplemental encapsulation of the melanoma-specific antigen tyrosinase-related protein 2 (TRP2). This melanoma-specific nanovaccine elicited a significant reduction of wild-type B16/F10 melanomas. Another focus was set on the characterization of the NC-induced immune response. The infiltration of different immune cells into tumor-draining lymph nodes and the tumor tissue in vivo was demonstrated. In particular, the anti-tumor immune response was shown to be mediated by antigen-specific activation of CD8+ cytotoxic T cells. In addition, the effect of NC treatment on the expression of immune checkpoint receptors by CD8+ T cells was investigated. The nanoparticle-based tumor vaccine presented in this PhD thesis can be flexibly adapted for personalized cancer therapies by encapsulation of patient-specific peptides and further modified with immune cell-addressing molecules.XI, 116 Seiten ; Illustrationen, Diagramm

    β2 Integrins on Dendritic Cells Modulate Cytokine Signaling and Inflammation-Associated Gene Expression, and Are Required for Induction of Autoimmune Encephalomyelitis

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    Heterodimeric β2 integrin surface receptors (CD11a-d/CD18) are specifically expressed by leukocytes that contribute to pathogen uptake, cell migration, immunological synapse formation and cell signaling. In humans, the loss of CD18 expression results in leukocyte adhesion deficiency syndrome (LAD-)1, largely characterized by recurrent severe infections. All available mouse models display the constitutive and ubiquitous knockout of either α or the common β2 (CD18) subunit, which hampers the analysis of the cell type-specific role of β2 integrins in vivo. To overcome this limitation, we generated a CD18 gene floxed mouse strain. Offspring generated from crossing with CD11c-Cre mice displayed the efficient knockdown of β2 integrins, specifically in dendritic cells (DCs). Stimulated β2-integrin-deficient splenic DCs showed enhanced cytokine production and the concomitantly elevated activity of signal transducers and activators of transcription (STAT) 1, 3 and 5, as well as the impaired expression of suppressor of cytokine signaling (SOCS) 2–6 as assessed in bone marrow-derived (BM) DCs. Paradoxically, these BMDCs also showed the attenuated expression of genes involved in inflammatory signaling. In line, in experimental autoimmune encephalomyelitis mice with a conditional DC-specific β2 integrin knockdown presented with a delayed onset and milder course of disease, associated with lower frequencies of T helper cell populations (Th)1/Th17 in the inflamed spinal cord. Altogether, our mouse model may prove to be a valuable tool to study the leukocyte-specific functions of β2 integrins in vivo
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