(437c) Engineered Tolerogenic Dendritic Cells Generate Robust T Regulatory Cells

Natural immune tolerance to self-antigens is maintained via T regulatory cells (Tregs), which actively surveil peripheral tissues or instruct naïve T cells in the thymus to remove or promote anergy of antigen specific T and B cells. This makes Tregs attractive targets for autoimmune disease and transplantation, but endogenous differentiation of de novo Tregs or ex vivo differentiation and reintroduction of Tregs has not been successful to date. A new strategy involves generating tolerogenic antigen presenting cells (APCs), which can instruct naïve T cells to differentiate into Tregs; however, the types of immunosuppressant drugs used to generate tolerogenic APCs often triggers cell death or has off target effects on unrelated immune interactions. We have recently shown that combinations of both immunosuppressing and immune activating signals can induce longer lived tolerogenic APC populations that can effectively induce Tregs. Here, we focus on delivering optimized combinations of immunosuppressants and immune stimulants to dendritic cells (DCs), which are the primary APC for T cell instruction. We identified several novel combinations to generate more robust tolerogenic dendritic cells (tolDCs), which we referred to as engineered tolDCs or etolDCs, using a high throughput screen. Candidate combinations were then selected based on their ability to upregulate the tolDC markers IL-10, TGF-β, PD-L1, PD-L2 and CD73 and downregulate markers of mature DCs, CD80, CD86, TNFα and IL-6 when screened using murine bone marrow derived DCs (BMDCs). We show that etolDCs express suppressive cytokines and live longer than tolDCs induced using immunosuppressants alone and have a unique mRNA expression profile. When etolDCs are incubated with naïve T cells ex vivo we observe suppression in T cell activation and proliferation and increases in T regulatory cells in an antigen specific fashion. This method of tolDC generation presents an opportunity as a cell therapy for autoimmune diseases and transplant rejection.