Keywords

Human, immune system, dendritic cells, nanoparticle, t helper, immunomodulation

Abstract

In the last 150 years, tremendous headway has been made in our understanding of the human immune system. Pioneers in the field such as Paul Ehrlich, Elie Metchnikoff, Louis Pasteur, Robert Koch and Walter Reed carried out seminal studies that established the groundwork for our understanding of humoral and cellular immunity in humans. However, this direct line of evidence into human immunology was diverted in the mid-20th century with the adoption of a model which allowed for investigators to use a reductionist-approach with the promise to resolve immunity at a molecular level. This revolutionary model was the scientific commercialization of various inbred strains of mice. It seems inconceivable how a four-legged nocturnal rodent managed to become the focus of billions of dollars of research to improve our understanding of human immunity. Nevertheless, this strange surrogate for human immunity did provide us with major conceptual advances in areas, such as identification of dendritic cell population heterogeneity, T cell help for B cell antibody production, MHC-restricted recognition of virusinfected cells, and even the discovery of cell types like NKT cells. However, these prior advances have now been prefaced with decades-worth of disappointing, non-translational findings. The best examples of such disappointments are in murine models of autoimmunity, cancer immunotherapy, and vaccinology where numerous studies have revealed promising outcomes in mice but were met with failure or limited success upon translation into humans. We do not look at this as a failure of the murine model; rather we consider it a call to arms to innovate in vitro surrogates to examine human immunity when otherwise bound by ethical limitation from working directly in humans. To overcome these challenges, we developed a system to interrogate novel immunogens that begins by generating human dendritic cells (DCs), a cell type necessary to mounting a protective immune response. DCs for research and clinical applications are typically derived from purified blood monocytes that are cultured in a cocktail of cytokines for a week or more. Because it has been iv suggested that these cytokine-derived DCs may be deficient in some important immunological functions and might not accurately represent antigen presenting cell (APC) populations found under normal conditions in vivo, there is an interest in developing methods that permit the derivation of DCs in a more physiologically relevant manner in vitro. Here, we describe a simple and reliable technique for generating large numbers of highly purified DCs that is based on a one-way migration of blood monocytes through a layer of human umbilical vein endothelial cells (HUVECs) that are cultured to confluency in the upper chamber of a Transwell device. The resultant APCs, harvested from the lower Transwell chamber, resemble other cultured DC populations in their expression of major histocompatibility (MHC) and costimulatory molecules, ability to phagocytose protein antigens and capacity to trigger primary antigen-specific T cell responses. This technique offers several advantages over the standard method of in vitro cytokine-driven DC development, including: (1) the rapidity of this approach, as DC differentiation occurs in only 2 days, (2) the differentiation process itself, which is more akin to the development of DCs under physiologic conditions and (3) the cost effectiveness of the system, since no monocyte pre-selection is required and DC development occurs in the absence of expensive recombinant cytokines. Taken together, this approach allows for the exploration of novel immunogens utilizing a physiologically representative population of APCs enriched from circulating blood. The outbreak of the swine-origin H1N1 influenza in the spring of 2009 took epidemiologists, immunologists, and vaccinologists by surprise and galvanized a massive worldwide effort to produce millions of vaccine doses to protect against this single virus strain. Of particular concern was the apparent lack of pre-existing antibody capable of eliciting cross-protective immunity against this novel virus, which fueled fears this strain would trigger a particularly far-reaching and lethal pandemic. Given that disease caused by the swine-origin virus was far less severe than expected, we hypothesized v cellular immunity to cross-conserved T cell epitopes might have played a significant role in protecting against the pandemic H1N1 in the absence of cross-reactive humoral immunity. We collaborated with bioinformaticians to develop an immunoinformatics approach to predict CD4+ T cell epitopes conserved between the 2008-2009 seasonal H1N1 vaccine strain and pandemic H1N1 (A/California/04/2009) hemagglutinin proteins that could act as novel immunogens and function as potential vaccine candidates or compliments to current vaccine formulations. We examined these peptides using T cells from human donors not exposed to the pandemic virus demonstrating that pre-existing CD4+ T cells can elicit cross-reactive effector responses against the pandemic H1N1 virus. As well, we showed the computational tools created by our collaborators were 80-90% accurate in predicting CD4+ T cell epitopes and their HLA-DRB1-dependent response profiles in donors that were chosen at random for HLA haplotype. Combined, these results confirm the power of coupling immunoinformatics to define broadly reactive CD4+ T cell epitopes with a highly sensitive in vitro model to verify these in silico predictions as a means to understand human cellular immunity, including cross-protective responses, and to define CD4+ T cell epitopes for potential vaccination efforts against future influenza viruses and other pathogens. It is thought that utilizing highly conserved peptides as novel immunogens of cellular immunity for future vaccination strategies may require an adjuvant for efficacy. However, the FDA has approved the use of only two adjuvant compounds (Alum or MPL®) which may not be compatible or offer effective immune enhancement in novel vaccine preparations, thereby soliciting the need for novel adjuvants. Nanoparticles have since been a topic of adjuvant potential. Nanoparticles harbor great potential because they possess unique physicochemical properties compared to their larger counter parts as a result of quantum-size effects and their inherent large surface area to volume ratio. These physicochemical properties govern how a nanoparticle will behave in its environment. However, vi researchers have only just begun to catalogue the biological effect these properties illicit. Moreover, little is known about the interaction between the immune system and NPs. However, in light of the recent development in new adjuvants that involves composites and coatings of polymers, lipids, ligands, TLR agonist, the ability of a simple metal oxide nanopowder to effectively induce or couple immunomodulation would provide researchers a basic alternative to costly and complex adjuvant development. Considering the evidence suggesting NPs can act as immunopotentiators, we questioned whether these materials can act not only as innate adjuvants, but as novel immunogens to cellular immunity. To accomplish this, we under took a set of studies to investigate any nanoparticle size-induced effects using TiO2, one of the most widely manufactured nanoparticles, as a model. We explored titanium dioxide synthesized into its three most commonly nanoarchitectures: anatase (7-10 nm), rutile (15-20 nm), and nanotube (10-15 nm diameters, 70-150 nm length) in comparison to a micron-sized formulation. We used the fully human autologous MIMIC® immunological construct has been utilized as a predictive, nonanimal alternative to diagnose nanoparticle immunogenicity. Cumulatively, treatment with titanium dioxide nanoparticles in the MIMIC® system led to elevated levels of proinflammatory cytokines and increased maturation and expression of costimulatory molecules on dendritic cells. Additionally, these treatments effectively primed activation and proliferation of naïve CD4+ T cells in comparison to dendritic cells treated with titanium dioxide microspheres, characteristic of an in vivo inflammatory response, providing evidence of a size induced difference between the nano-sized and micron-sized material, revealing novel immune cell recognition and activation by a crystalline nanomaterial in a size-dependent manner. Having identified nanomaterial size as a contributing feature of nanoparticle induced immunopotentiation, we became interested if additional physicochemical properties such as surface vii reactivity or catalytic behavior could also be immunostimulatory. Moreover, because we witnessed a stimulatory effect to dendritic cells following nanoparticle treatment, we were curious how these nanoparticle-touched dendritic cells would impact adaptive immunity. Since TiO2 acts as an oxidant we chose an antioxidant nanoparticle, CeO2, as a counterpart to explore how divergent nanoparticle surface reactivity impacts innate and adaptive immunity. We focused on the effect these nanoparticles had on human dendritic cells and TH cells as a strategy towards defining their impact to cellular immunity. Combined, we report that TiO2 nanoparticles potentiate DC maturation inducing the secretion of IL- 12p70 and IL-1B, while treatment with CeO2 nanoparticles induced IL-10, a hallmark of suppression. When delivered to T cells alone TiO2 nanoparticles induced stronger proliferation in comparison to CeO2 which also stimulated TReg differentiation. When co-cultured in allogeneic T cell assays, the materials directed alternate TH polarization whereby TiO2 drives largely a TH1 dominate response, whereas CeO2 drove a TH2 bias. Combined, we report a novel immunomodulatory capacity of nanomaterials with catalytic activity. While unintentional exposure to these nanomaterials could pose a serious health risk, development and targeted use of such immunomodulatory nanoparticles could provide researchers with new tools for novel adjuvant strategies or therapeutics.

Notes

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Graduation Date

2012

Semester

Summer

Advisor

Self, William

Degree

Doctor of Philosophy (Ph.D.)

College

College of Medicine

Department

Molecular Biology and Microbiology

Degree Program

Biomedical Sciences

Format

application/pdf

Identifier

CFE0004629

URL

http://purl.fcla.edu/fcla/etd/CFE0004629

Language

English

Release Date

February 2018

Length of Campus-only Access

5 years

Access Status

Doctoral Dissertation (Campus-only Access)

Subjects

Dissertations, Academic -- Medicine, Medicine -- Dissertations, Academic

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