Sustained microparticle-mediated intracellular immunomodulation enhances tolerogenic dendritic cell stability and antigen-specific Treg induction.

Tolerogenic dendritic cells (tolDCs) are a promising cell therapy platform for restoring immune tolerance, but their clinical efficacy is limited by poor phenotypic stability and short in vivo persistence following transfer. Here, we developed biodegradable poly(lactic-co-glycolic acid) (PLGA) microparticles co-loaded with ovalbumin (OVA) and a combinatorial Push/Pull Immunomodulation (PPI) formulation consisting of the TLR7/8 agonist resiquimod (R848) and the JAK2 inhibitor CEP-33779. We hypothesized that sustained intracellular release of immunomodulators from phagocytosed microparticles would stabilize tolDC phenotype and enhance antigen-specific regulatory T cell (Treg) responses. PPI microparticles (PPI MPs) demonstrated sustained cargo release over 30 days, with the most pronounced pH sensitivity observed for CEP-33779, and were efficiently internalized by murine bone marrow-derived dendritic cells (BMDCs) and human monocyte-derived dendritic cells. BMDCs loaded with PPI MPs exhibited prolonged viability, increased PD-L1 and IL-10 expression, and resistance to inflammatory reprogramming following lipopolysaccharide challenge compared to dexamethasone-loaded microparticles or soluble drug controls. Administration of PPI MPs during BMDC differentiation further enhanced tolerogenic programming, producing marked increases in PD-L1 and IL-10 while suppressing TNFα secretion. Following adoptive transfer in vivo, PPI MP-treated DCs demonstrated prolonged persistence and accumulation in draining lymph nodes for up to 7 days. Finally, PPI MP-treated DCs induced robust antigen-specific Foxp3+ regulatory T cell responses in OVA-sensitized mice and suppressed antigen recall responses ex vivo. Together, these findings demonstrate that sustained intracellular combinatorial immunomodulation can generate a durable and functionally stable tolDC phenotype with enhanced antigen-specific tolerogenic activity, supporting this platform as a potential strategy for autoimmune therapies.