Regulation of Immune Responses by Genetically-Engineered Dendritic Cells and Exosomes

Approximately 5-8% of Americans are affected by some type of autoimmune disease. The current standard of care for these patients involves pharmacologic therapy to induce systemic immunosuppression, which carries significant side effects. There is a need for therapies that effectively restore immune-mediated tolerance towards the body, but do not cause inappropriate immunosupression.The following dissertation details the synthesis, characterization, and performance of tolerogenic dendritic cells (DC), as well as the exosomes that they secrete. Exosomes are small (40-100 nm) membrane-bound microvesicles produced by reverse budding of the membrane of the multivesicular endosome in DC, as well as many other cell types. DC were transduced with adenoviral vectors expressing target genes in order to manipulate their function. After transduction, DC and their exosomes were examined for the ability to modulate disease-induced inflammation in the NOD mouse model of type 1 diabetes and the delayed-type hypersensitivity (DTH) model.DC transduced with an IL-4 expressing vector are capable or preventing the onset of hyperglycemia when administered to 12-weeks-old and 12-16 week-old prediabetic mice. Treated mice demonstrate modulation of T-cell mediated β-cell autoimmunity, including reduced insulitis, increased FoxP3 expression in the islet-draining lymph nodes, and Th2 skewing of islet-antigen induced cytokine secretion profile. We further demonstrate that transduction of DC with adenoviral vectors expressing indoleamine 2,3-diooxygenase (IDO) and CTLA-4-Ig are efficient means to generate IDO+ DC in vitro, and that these DC effectively reduce paw swelling in the DTH model of antigen-specific inflammation. The exosomes secreted by these IDO+ DC contain IDO protein, and suppress DTH responses at a level comparable to their parental DC in a manner dependent on B7-1 and B7-2. To confirm the role of B7 costimulatory molecules on tolerogenic DC and exosomes, we further demonstrate that B7-1 and B7-2, but not PD-L1 or PD-L2, are required for in vivo suppressive activity of rIL-10 treated DC and their secreted exosomes.This endeavor has increased the knowledge regarding therapeutic DC and exosomes. We have demonstrated strategies to generate tolerogenic DC and exosomes, as well as factors that are required for their activity in vivo