Synergistic immunomodulatory combinations induce robust human tolerogenic dendritic cells for antigen-specific regulatory T cell generation.

Restoring antigen-specific immune tolerance remains a central challenge in the treatment of autoimmune diseases, as conventional immunosuppressive therapies lack specificity and can compromise protective immunity. Tolerogenic dendritic cell (tolDC) therapies offer a promising strategy for inducing durable, antigen-specific regulatory T cell (Treg) responses, but current methods for generating tolDCs are limited by poor longevity and Treg generative capacity. This study evaluates the clinical potential of Push/Pull Immunomodulation (PPI), a novel combinatorial approach identified through high-throughput molecular screening, in human monocyte-derived dendritic cells (moDCs). The phenotype, cytokine profile, and longevity of PPI-treated moDCs were benchmarked against conventional tolDC induction agents. Functional assays assessed the capacity of PPI-tolDCs to induce antigen-specific Tregs. PPI-treated moDCs exhibited increased expression of tolerogenic markers (interleukin-10, programmed cell death receptor ligand 1, and B- and T-lymphocyte attenuator), enhanced in vitro longevity, and a unique transcriptomic signature characterized by upregulation of IDO1, IDO2, and T cell-sustaining cytokines. Notably, PPI9 induced robust, antigen-specific Treg responses, suggesting the potential for long-term tolerance induction. Although transient tumor necrosis factor-α release and moderate upregulation of CD40 and CD86 were observed, these did not impair Treg generation, indicating that PPI-tolDCs overcome key barriers associated with conventional tolDC therapies. PPI enables the generation of stable, long-lived human tolDCs with superior capacity to induce antigen-specific Tregs. Ongoing studies will further explore the translational potential of PPI-tolDCs for clinical applications in autoimmunity and transplantation. SIGNIFICANCE STATEMENT: This study validates a novel molecular combination to generate tolerogenic dendritic cells with primary human cells. These tolerogenic dendritic cells have potential use as a cell therapy for autoimmune diseases and transplantation.