Authors:	Shen, Limei
Title:	The role of dendritic cells (DC) in Friend retrovirus-induced immunosuppression and immunotherapeutic applications of functionalized nanoparticles
Online publication date:	17-Jan-2014
Year of first publication:	2014
Language:	english
Abstract:	Friend murine leukemia Virus (FV) infection of immunocompetent mice is a well- established model to acquire further knowledge about viral immune suppression mechanisms, with the aim to develop therapeutics against retrovirus-induced diseases. Interestingly, BALB/c mice are infected by low doses of FV and die from FV-induced erythroleukemia, while C57/BL6 mice are infected by FV only at high viral dose, and remain persistently infected for their whole life. Due to the central role of dendritic cells (DC) in the induction of anti-viral responses, we asked for their functional role in the genotype-dependent sensitivity towards FV infection. In my PhD study I showed that bone marrow (BM)-derived DC differentiated from FV-infected BM cells obtained from FV-inoculated BALB/c (FV susceptible) and C57BL/6 (FV resistant) mice showed an increased endocytotic activity and lowered expression of MHCII and of costimulatory receptors as compared with non-infected control BMDC. FV-infected BMDC from either mouse strain were partially resistant towards stimulation-induced upregulation of MHCII and costimulators, and accordingly were poor T cell stimulators in vitro and in vivo. In addition, FV-infected BMDC displayed an altered expression profile of proinflammator cytokines and favoured Th2 polarization. Ongoing work is focussed on elucidating the functional role of proteins identified as differentially expressed in FV-infected DC in a genotype-dependent manner, which therefore may contribute to the differential course of FV infection in vivo in BALB/c versus C57BL/6 mice. So far, more than 300 proteins have been identified which are differently regulated in FV-infected vs. uninfected DC from both mouse strains. One of these proteins, S100A9, was strongly upregulated specifically in BMDC derived from FV-infected C57BL/6 BM cells. S100A9-/- mice were more sensitive towards inoculation with FV than corresponding wild type (WT) mice (both C57BL/6 background), which suggests a decisive role of this factor for anti-viral defense. In addition, FV-infected S100A9-/- BMDC showed lower motility than WT DC. The future work is aimed to further elucidate the functional importance of S100A9 for DC functions. To exploit the potential of DC for immunotherapeutic applications, in another project of this PhD study the usability of different types of functionalized nanoparticles , based on either dextran molecules or a ferromagnetic solid core, to induce potent immune responses was assessed. We tested nano-sized dextran (DEX) particles to serve as a DC-addressing nanocarrier platform. Non-functionalized DEX particles had no immunomodulatory effect on bone marrow (BM)-derived DCin vitro. However, when adsorbed with ovalbumine (OVA), DEX particles were efficiently engulfed by DC in a mannose receptor-dependent manner. A DEX-based nanovaccine containing OVA and lipopolysaccharide (LPS) as a DC stimulus induced strong OVA peptide-specific CD4+ and CD8+ T cell proliferation both in vitro and in vivo, as well as a robust OVA-specific humoral immune response (IgG1>IgG2a) in vivo. Accordingly, this nanovaccine raised a stronger induction of cytotoxic CD8+ T cells than obtained upon administration of OVA and LPS in soluble form. Therefore, DEX-based nanoparticles constitute a potent, versatile and easy to prepare nanovaccine platform for immunotherapeutic approaches. In order to enhance tumor antigen-specific immune responses by in vivo delivery of antigen and adjuvant specifically to DC, three issues were considered for the ferromagnetic solid core nanoparticle: Due to the inherent capability of CD8+DEC205+ DC to efficiently cross-present antigens and thereby prime CD8+ T cells, solid core nanoparticles (NP) were conjugated with a DEC205-specific antibody (αDEC205) In addition, NP were coated with the model antigen ovalbumin (OVA), constitutively expressed by a OVA-transduced B16 melanoma subline (B16/OVA) used for subcutaneous tumor inoculation. NPs were coupled in addition with the TLR9 ligand CpG as an adjuvant to activate DC. In vivo studies revealed superior efficacy of this trifunctional NP formulation (NP[OVA+CpG+αDEC205]) to evoke antigen-specific T cell (CD4+,CD8+) proliferation, and induction of cytotoxic T lymphocyte responses, as compared with other types of NPs (NP[OVA], NP[OVA+CpG]). Accordingly, in a therapeutic B16/OVA melanoma model, only tumor-burdened mice vaccinated with trifunctional NP showed a pronounced anti-tumor response as reflected by an arrested tumor growth and significantly higher survival rate as compared with groups of mice left untreated or vaccinated with either of the other NP formulations. Interestingly, in vivo these solid core NP were found to bind specifically to B cells due to opsonization with heat-labile serum components as confirmed by in vitro studies. Furthermore, NP that codelivered with ovalbumin (OVA) and CpG mounted OVA-specific antibody production. Additional conjugation with aDEC205 antibody, known to enhance antigen uptake by dendritic cells (DC) and subsequent induction of T cell helper cells (see above) which provide B cell activation in an antigen-specific manner, indeed enhanced OVA-specific antibody production, with a strong Th1 bias. Therefore, the efficacy of these Fe-NP for the B cell based immunotherapy was analysed. In therapeutic OVA-based anaphylaxis model, the particles conjugated with OVA and CpG inhibited significant IgE production, and the survival in the group which immunized with p(OVA-CpG) and p(OVA-CpG-aDEC205) was increased. In a model of OVA-based acute asthma, administration of NP conjugated with OVA+CpG was effective to attenuate bronchial hyper responsiveness, and inflammation of the lung was reduced. Taken together, Fe-NP nanoparticles constitute a well available nanoform most suitable for the induction of strong celluler and humoral immune responses, essential for the therapy of infectious diseases and supportive of anti-tumor.
DDC:	500 Naturwissenschaften 500 Natural sciences and mathematics
Institution:	Johannes Gutenberg-Universität Mainz
Department:	FB 04 Medizin
Place:	Mainz
ROR:	https://ror.org/023b0x485
DOI:	http://doi.org/10.25358/openscience-3663
URN:	urn:nbn:de:hebis:77-36214
Version:	Original work
Publication type:	Dissertation
License:	In Copyright
Information on rights of use:	https://rightsstatements.org/vocab/InC/1.0/
Extent:	183 S.
Appears in collections:	JGU-Publikationen