(530e) Combination of Toll-like-Receptor Agonists and Inhibitors for Generation of Antigen Specific Tolerance

Autoimmune diseases are a growing problem in the western world and are particularly difficult to treat due to the wide range of disease pathogenesis and symptoms. As a result, treatments typically include broad immune suppression that leaves patients vulnerable for other infections. The project describes a new versatile method for autoimmune disease therapy by using toll-like-receptor (TLR) agonist and inhibitor combinations to generate long lasting antigen specific T regulatory (Treg) responses. Autoimmune diseases originate with self-reactive antigens or self-reactive mimetics being uptaken and presented on antigen presenting cells (APC) and activating downstream adaptive immune cells leading to either a B or T cell or both. Under normal physiological conditions, this antigen presentation occurs in primary lymphoid organs and is accompanied with various inhibitory and activating signals to generate a Treg response that actively depletes any auto-reactive adaptive immune response to the self-antigen in question. Here, we seek to mimic this combination of positive and negative signaling generated in peripheral tolerance to induce auto-antigen display on tolerogenic dendritic cells (tolDCs). TolDCs are unique phenotype of the primary antigen presenting cells (APC), which expresses high levels of PD-L1 and IL-10, tolerizing markers that facilitate Treg development. We screened over 15,000 combinations of TLR inhibitors and agonists using an APC cell line to identify a small number of TLR agonist/inhibitor combinations that reduce both NF-kB and IRF transcription factor signaling, additional hallmarks of tolDCs. Using secondary screens, identified a small library of combinations that induces tolDC phenotypes from naïve bone marrow derived dendritic cells (BMDCs). After identifying optimal candidates, we will develop a liposomal formulation of the inhibitors with a model antigen, OVA, and the MOG peptide (an autoantigen for a murine model of multiple sclerosis). We already have preliminary data demonstrating that TLR agonist/inhibitors can generate tolDCs and antigen specific Tregs in these models using a TLR agonist/inhibitor combination identified from a much smaller library. The preliminary liposome formulation was tested in vivo and triggered a two-fold increase in tolerogenic DCs (CD11c+, CD80-, PD-L1+) and a two-fold increase of OVA-specific Tregs (CD4+, CD25+, FoxP3+, OVA-Tetramer+). Furthermore, there was a complete reduction in any OVA specific antibodies or CD8 T cells when compared to non-inhibitor loaded controls. This is strong evidence that liposome formulations of TLR agonists/inhibitors can generate antigen specific T reg responses that can functionally reduce autoimmune responses.