Thesis (Ph.D.)--Victoria University of Wellington, 2009. / Includes bibliographical references.

<p>Despite aggressive surgical resection, cytotoxic chemotherapy, and external beam radiotherapy, most cases of glioblastoma (GBM) remain recalcitrant. These outcomes necessitate novel developmental therapeutics that spare normal tissue. Immunotherapy is a promising novel adjuvant treatment that can harness the cytotoxic capacity of the immune system against tumor-associated antigens with exquisite specificity. To circumvent the challenges associated with the advancement of adoptive cellular immunotherapy, we developed a novel treatment platform, which leverages the use of commercially available and clinically translatable nanoparticles (NPs) that can be combined with tumor derived RNA to peripherally activate T cells against GBM antigens. Although cancer vaccines have suffered from weak immunogenicity, we have advanced a NP vaccine formulation that can reshape a host’s immune profile through combinatorial delivery of RNAs encoding for tumor antigens and RNAs encoding for immunomodulatory molecules to mediate long-lived T cell persistence. </p><p>We sought to assess if vaccination with amplified tumor derived RNA encapsulated in lipophilic NPs could be assembled to transfect antigen presenting cells (APCs) in vivo and induce therapeutic anti-tumor immunity in pre-clinical murine tumor models. We hypothesized that RNA encapsulated nanoliposomes would localize to reticuloendothelial organs such as the spleen and liver, transfect APCs therein and induce peripheral antigen specific T cell immunity against GBM. Since activated T cells can cross the blood brain barrier and exert their effector functions against GBM antigens, peripheral transfection of APCs by RNA-NPs represents an attractive vaccination approach for priming endogenous immunity against refractory brain tumors.</p><p>We screened several translatable NP formulations for their ability to transfect dendritic cells (DCs) in vitro with GFP mRNA. We demonstrated that the NP DOTAP was the most promising translatable formulation compared to alternative cationic liposomal preparations and linear polyethylenimine NPs with and without DC targeting mannose receptors. RNA-NP vaccines formulated in DOTAP were shown to induce in vivo gene expression and preserve RNA stability over time. We determined that intravenous (IV) injection of RNA-NPs was requisite for inducing functional antigen specific immunity, which was superior to standard peptide vaccines formulated in complete Freund’s adjuvant (CFA). IV administered RNA-NPs localized to splenic and hepatic white blood cells (WBCs); these cells expanded antigen specific T cells when transferred to naïve immunocompetent mice. RNA-NPs induced increased percentages of B7 co-stimulatory molecules, but also elicited compensatory PD-L1 expression. We enhanced the immunogenicity and anti-tumor efficacy of RNA-NP vaccines by combining RNA-NPs with immune checkpoint blockade against PD-L1. We also enhanced the immunogenicity and efficacy of this platform by simply combining mRNAs encoding for immunomodulatory cytokines (i.e. GM-CSF). Finally, we demonstrated that RNA-NP vaccines mediate anti-tumor efficacy against intracranial and subcutaneous melanomas and engender therapeutic anti-tumor efficacy in a cellular immunotherapy model against a radiation/temozolomide resistant invasive murine high-grade glioma.</p><p>GBM remains invariably associated with poor patient outcomes thus necessitating development of more targeted therapeutics. Clinically translatable RNA-NPs form stable complexes making them amenable to overnight shipping. They induce potent immune responses when administered systemically and mediate robust anti-tumor efficacy that can be enhanced through co-delivery of immunomodulatory RNAs. </p><p>This technology can simultaneously bypass the complexity of cellular therapeutics while cutting down the time to generation of personalized vaccines. Since RNA-NP vaccines can be made within days from a tumor biopsy, providing near immediate immune induction against GBM, these formulations can provide a more feasible and effective therapy with a wide range of applicability for all malignancies that can be targeted using RNA obtained from surgical resection of solid tumors.</p> / Dissertation

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Lung cancer is the leading cause of cancer mortality worldwide. Understanding biological processes of specific antitumor immune response remains of an eminent interest and represents necessity for designing successful antitumor immunotherapeutic strategies. The theoretical part of the thesis describes components of the immune system that are involved in antitumor response and discusses their role in the hitherto known and used lung cancer immunotherapy. In the practical part of the thesis, two studies studying different aspects of anticancer immune response are described. Both studies were conducted in cooperation with 3rd Surgical Department 1st Faculty of Medicine, Charles University and University Hospital Motol and with the biotechnology company Sotio a.s. The first study is focused on the humoral component of the specific antitumor response and prospectively analyses serum frequency of antitumor antibodies against NY-ESO-1, Her2/neu and MAGE-A4 antigens in 121 patients with NSCLC. Here it was shown for the first time that tobacco smoking significantly increases the frequency of NY- ESO-1 antibodies in sera of smokers in comparison to ex-smokers and non-smokers. The second study is focused on the cellular component of the specific antitumor response investigating the activity of the dendritic...

BACKGROUND: Previous work has shown that follicular dendritic cells (FDCs) play an important role in selecting B cells such that antigens are responded to in a specific manner. FcγRΙΙB (CD32) is an antibody constant-region receptor found on FDCs and mutation of this receptor in humans is associated with Systemic Lupus Erythematosus (SLE). In addition, previous work has demonstrated that autoreactive germinal centers are the product of expression of interferon alpha (ΙFNα) by FDCs, so FcγRIIB signaling may involve modulation of IFNα signaling. OBJECTIVE: Because FcγRIIB mutation is associated with SLE and FDCs have been shown to be important in orchestrating B cell responses, understanding FcγRIIB on FDCs helps characterize B cell repertoire development in response to antigen—whether the antigen is foreign or self, as is the case in autoimmunity. Better characterization of the role of FcγRIIB could have consequences for autoimmune and cancer therapy. This study seeks to determine the role of FcγRΙΙB on FDCs in germinal center B cell selection dynamics within single, autoreactive germinal centers. METHODS: This study compares transplanted wild-type (B6) B cells—that are driven to be autoimmune by simultaneously transplanted autoimmune B cells—in two stromal cell settings: first, in germinal centers containing wild-type FDCs and second, in germinal centers containing FcγRIIB-knockout FDCs. Transplanted B6 B cell populations express photoactivatable protein, which allows for sorting of B cells from individual germinal centers. B cell sequences from single germinal centers were analyzed to determine how focused each germinal center response was and how the B cells differ in maturity and affinity for antigen. Finally, mice expressing a lineage-tracing system were treated with IFNα in order to observe the cytokine’s effect on B cell selection. RESULTS: Cells sorted from germinal centers containing FcγRIIB-knockout FDCs contain a distinguished population of less-developed B cells, as quantified by population-based analysis of their variable heavy chain genes. Overall, the IgM sequences from B cells sorted from germinal centers (GCs) containing FcγRIIB-knockout follicular dendritic cells displayed lower levels of somatic hypermutation (SHM) (p<.05) and shorter hypervariable regions (CDR3) (p<.05) compared to other B cell populations. Values computed to summarize how many different B cell lineages were present in a GC—its “clonality”—did not vary between the two mouse populations, although FcγRIIB-knockout FDC germinal centers displayed a correlation between clonality and immunoglobulin (Ig) isotype expression (R2= .85). Finally, lineage tracing mice receiving injections of interferon alpha (IFNα) displayed no difference in GC clonality compared to those receiving vehicle and assays of IFNα downstream signaling genes also displayed no change. CONCLUSIONS: FcγRIIB encourages more stringent selection of immature B cells in germinal centers as evidenced by survival of less developed B cells as defined by degree of somatic hypermutation and CDR3 length in GCs comprising FcγRIIB-knockout FDCs. In spite of this, sequence-based measures of germinal center clonality as completed here may fail to capture the functional results of B cell selection. In addition, the link between FcγRIIB and IFNα requires further investigation. / 2019-07-03T00:00:00Z

Interleukin-10 (IL-10) deficient dendritic cells (DCs) are potent antigen presenting cells during Chlamydia infection. To further understand the mechanism underlying this protective property in IL-10 DCs, we combined two proteomic techniques: 2DIGE and liquid chromatography mass spectrometry. We then performed western blotting on proteins from Chlamydia infected wild type (WT) and IL-10 knock out (KO) DCs. The results showed that alpha enolase (ENO1), a metabolic enzyme involved in the ninth step of glycolysis, was significantly upregulated in Chlamydia infected IL-10KO DCs compared to WT DCs. We further studied the role of ENO1 by silencing ENO1 gene using lentiviral siRNA technology. Flow cytometry, confocal microscopy, cytokine analysis, infectivity analysis and T-cell proliferation analysis were also used to determine DC function. We then analyzed the effect of the ENO1 knockdown on DC metabolism during Chlamydia stimulation. The results showed that DC maturation and activation were decreased in ENO1 knockdown DCs that were stimulated with Chlamydia compared to the Chlamydia stimulated WT DCs. In addition, pyruvate concentration decreased significantly in ENO1 knockdown DCs stimulated with Chlamydia. The mitochondrial structure of Chlamydia stimulated ENO1 knockdown DCs appeared damaged compared to the Chlamydia stimulated WT DCs. The function of ENO1 in the immune response to Chlamydia trachomatis infection is unknown. However, the results from this study indicates that ENO1 may contribute to DC metabolism and mitochondrial homeostasis which may play a role in the maturation and antigen presenting function of DCs.

Saliva of Ixodid ticks contains a whole array of pharmacologically active molecules with vasodilatory, antihemostatic, and immunomodulatory activities. This thesis focuses on two types of salivary proteins, serpins and cystatins, and their role in immunomodulation. These protease inhibitors are known to affect many biological functions. To better understand their role in tick saliva we examined their effect on dendritic cells and their ability to modulate the immune response after pathogen infection. As model pathogens, Borrelia spirochetes and tick-borne encephalitis virus were used.

In this study we examined the effect of the tick saliva from I. ricinus and the effect of recombinant protein IRS-2 from the saliva of I. ricinus on dendritic cells derived from the mice bone marrow. We studied their effect on the production of cytokines by dendritic cells after the stimulation by B. burgdorferi, their effect on the expression of genes, that participate in phagocytosis, and the impact of the tick saliva on phagocytosis of B. burgdorferi by dendritic cells.

Submitted by Ana Maria Fiscina Sampaio (fiscina@bahia.fiocruz.br) on 2014-02-07T18:23:30Z No. of bitstreams: 1 Renata Siqueira Portella.. Presença de células....pdf: 2025004 bytes, checksum: a85d3195f20f2a79bd6ff1b32049c260 (MD5) / Made available in DSpace on 2014-02-07T18:23:30Z (GMT). No. of bitstreams: 1 Renata Siqueira Portella.. Presença de células....pdf: 2025004 bytes, checksum: a85d3195f20f2a79bd6ff1b32049c260 (MD5) Previous issue date: 2013 / Fundação Oswaldo Cruz. Centro de Pesquisa Gonçalo Moniz. Salvador, BA, Brasil / Universidade Federal da Bahia. Faculdade de Medicina. Salvador, BA, Brasil / A doença de Chagas é caracterizada por apresentar duas fases com curso clínico bastante variável. Na fase aguda ocorre uma intensa miocardite, sendo o parasita facilmente detectado no sangue periférico e nos tecidos. A fase crônica cardíaca é caracterizada por uma cardiopatia, com intensa destruição das fibras cardíacas, presença de áreas de fibrose e escassos parasitas. Os mecanismos envolvidos na patogenia dessa miocardite ainda não são muito claros. Acredita-se que as células reguladoras e as células dendríticas estejam envolvidas nesse processo. Para compreender os mecanismos envolvidos, na resposta inflamatória à infecção pelo T. cruzi, resolvemos investigar a participação das células dendríticas, das células reguladoras e o perfil dos linfócitos T CD4+ e CD8+, utilizando duas linhagens de camundongos isogênicos, que apresentam diferentes graus de susceptibilidade a infecção. Em nossos resultados constatamos que os camundongos DBA/1 apresentaram maior sobrevida à fase aguda (90%), mesmo tratando os camundongos A (70%) com benzonidazol por três dias consecutivos, com o intuito de diminuir a carga parasitária prevenindo a alta mortalidade. A resistência dos DBA/1 e a susceptibilidade dos A, a infecção pelo T. cruzi, estaria relacionada ao perfil da resposta inflamatória e regulatória desenvolvida no decorrer da doença. Observamos que os camundongos DBA/1 possuem mais células dendríticas ativadas no baço e coração e mais células T CD4+CD25hi do que os camundongos da linhagem A. Essa diferença do perfil de resposta pode estar provocando uma maior expansão e diferenciação dos linfócitos T CD4+ pelas células dendríticas, levando ao controle da carga parasitaria. / Chagas’diseasse, due to Trypanosoma cruzi infection is characterized by the development of two phases with a variable clinical course. During the acute phase there occurs a severe myocarditis with the parasite being easily detected in peripheral blood and tissues. The chronic cardiac phase is characterized as a chronic cardiopathy, when severe destruction of cardiac myocells and fibrosis are present and parasites are rare. The mechanisms involved in the pathogenesis of this myocarditis are somewhat obscure. It is believed that regulatory and dendritic cells play a role in this process. In an attempt to clarify the mechanisms involved in the inflammatory response to infection with T. cruzi we decided to investigate the participation of dendritic and regulatory cells and of TCD4 and TCD8 lymphocytes, using two strains of isogenic mice , which exhibit different degrees of susceptibility to infection. Our results have shown that the DBA/1 mice presented a higher survival (90%) in the acute phase than the A mice (70%), even when these were treated with Benznidazole for three consecutive days, with the objective of to reduce the parasitemia and the high mortality. Resistance of DBA/1 mice and susceptibility of A mice could be related to the evolution of the inflammatory and regulatory responses, during the infection. It was seen that DBA/1 mice disclosed a higher number of activated dendritic cells in the spleen and heart and a higher number of T CD4+CD25hi than the mice of A strain. This differences of the response could be influencing in the TCD4 differentiation and on the control of parasitic load