(23d) Advanced Immunomodulatory Strategies for Tolerogenic Dendritic Cell Induction in Autoimmune Therapy

T regulatory cells (Tregs) play a crucial role in natural immune tolerance towards self-antigens by monitoring peripheral tissues or guiding naïve T cells within the thymus to eliminate or render antigen-specific T and B cells anergic. This positions Tregs as potential therapeutic agents for autoimmune diseases and transplant procedures. However, the internal generation of new Tregs or their differentiation outside the body and subsequent reintroduction has not proven effective so far. A novel approach entails the creation of tolerogenic antigen-presenting cells (APCs) capable of directing naïve T cells towards becoming Tregs. Nonetheless, the immunosuppressive drugs traditionally employed to produce tolerogenic APCs often lead to cell death or unintended impacts on non-targeted immune functions. Our research introduces an innovative approach through the application of Push/Pull Immunomodulation (PPI) — a novel methodology that combines toll-like receptor agonists and immunosuppressants, identified through extensive high-throughput screening. The aim is to generate tolerogenic dendritic cells (tolDCs), with the goal of recovering immune tolerance at a foundational level. Through high-throughput screening and optimization, we identified top three PPI formulations including PPI 1, PPI 7 and PPI 9 which are capable of generating effective tolerogenic dendritic cells (tolDCs) from BMDCs ex vivo. Moreover, we demonstrated that these optimized PPI-based tolDCs are able to introduce antigen-specific Tregs. We also tested the top three PPI formulations in monocytes from human peripheral blood mononuclear cells (PBMC). The study included a detailed investigation of seven different treatment methodologies alongside our top three PPI-treated groups. Our research focused on the expression of key tolerogenic markers (e.g.,PD-L1, BTLA, HLA-DR, CD40, CD1c, CD86) through flow cytometry and cytokine secretion analysis via ELISA. Additionally, we did a bulk mRNA sequencing to compare gene expression profiles among naturally arising tolDCs (ntolDCs), induced tolDCs (itolDCs), and PPI-tolDCs. We revealed that our PPI-tolDCs exhibit increased levels of inhibitory cytokines and have a longer survival time than tolDCs created with only immunosuppressants, and display a distinct mRNA expression pattern. This technique of generating tolDCs offers a novel cell therapy pathway for managing autoimmune conditions and mitigating transplant rejection.