Phosphatidylserine (PS) is the most abundant negatively charged phospholipid in the cell membrane. Despite comprising 12% of total phospholipid and being restricted primarily in the inner leaflet of the membrane bilayer, the consequences of PS externalization to the outer leaflet is biologically significant, especially in hemostasis and apoptosis. The exposure of PS on the surface of apoptotic cells functions as an "eat me" signal that initiates macrophage recognition, uptake, and removal of the apoptotic debris. The apoptotic process is an immunologically silent event, as these apoptotic cells are cleared without activation of the immune system. There is considerable literature evidence that support the fact that PS is a critical mediator in apoptosis, facilitating the efficient removal of cell debris in order to maintain tolerance to self-proteins by immunosuppressive mechanisms and tissue homeostasis.

The key function of PS exposure in apoptosis could be exploited for therapeutic implications, such as mitigation of immunogenicity towards therapeutic proteins. The focus of our laboratory is the development of tolerogenic liposomal formulations to reduce immunogenicity and induce immunological hypo-responsiveness towards therapeutic proteins. Factor VIII (FVIII) served as the model protein, as the dysfunction or deficiency of FVIII manifests in Hemophilia A (HA). Despite enzyme replacement therapy as the first line of therapy, about 30% of severe HA patients develop neutralizing (NAbs) or inhibitory titers, severely complicating efficacy of therapy. As a result, any approach designed to reduce immunogenicity and induce tolerance would address an unmet clinical need. Previous studies have shown that subcutaneous pre-treatment of FVIII in the presence of PS liposomes induced hypo-responsiveness after subsequent rechallenge to free FVIII in HA mice. The PS-mediated mechanism involves induction of tolerogenic dendritic cells, secretion of regulatory cytokine TGF-beta, generation of regulatory T cells, inhibition of memory B cell development, and reduction of antibody production, resulting in the property of PS to convert an immunogen to a tolerogen. The overall goal of this dissertation is to extend the application of our PS tolerance strategy into multiple sclerosis, a model disease for autoimmunity, as well as to develop and optimize this strategy to enhance the tolerogenic effects of PS tolerance induction.

Chapter 1 provides an overview of the biological functions of PS. The involvement of PS in hemostasis and apoptosis are discussed along with the contribution of receptors and structural modifications that may influence the biological outcome of PS exposure. We then highlight how the understanding of PS mechanism can be exploited for therapeutic applications, such as in viral infection, cancer, autoimmunity, and mitigation of immunogenicity. Further, we highlight the property of PS conversion of an immunogen to a tolerogen through our previous work and how that property could be exploited to induce immunological tolerance towards a protein.

In Chapter 2, we investigated whether PS liposomes can utilized with multiple antigens other than FVIII. We found that pre-exposure of acid alpha glucosidase (GAA) in the presence of PS can induce a durable and long-lasting hypo-responsiveness in Pompe Disease mice. In addition, we demonstrated that the PS receptor, T-cell immunoglobulin- and mucin 4 (TIM-4) receptor, is involved in PS-mediated hypo-responsiveness. Finally, we showed that pre-exposure of myelin oligodendrocyte glycoprotein (MOG) peptide, a known auto-antigen for multiple sclerosis (MS), in the presence of PS can alter the disease onset and disease severity of experimental autoimmune encephalomyelitis (EAE), a murine model for human MS. We further determined that an increase in CD4 +FoxP3+ regulatory T cell expression was exhibited in mice that were pre-treated with PS MOG35-55, suggesting the involvement of regulatory T cells in PS-mediated effects in EAE.

After demonstrating that PS can be utilized to induce tolerance towards therapeutic proteins and can be extended to re-tolerize the immune system to a self-protein in autoimmune conditions, we sought to optimize the PS particle in Chapter 3 in order to improve the translatability of this strategy into the clinic and enhance its tolerogenic properties. The identification of a specific structural PS species that could be responsible for the PS ability to convert an immunogen to a tolerogen is essential in order to develop a homogeneous formulation for successful translation. (Abstract shortened by ProQuest.).