PLGA microparticle combination therapy induces a tolerogenic phenotype in human monocyte-derived dendritic cells

摘要

Introduction: Type 1 diabetes (T1D) is an autoimmune disease characterized by the loss of immune tolerance to insulin-producing pancreatic beta cells, followed by their T cell-mediated destruction. A major therapeutic goal is the induction of lasting T cell tolerance to beta cell antigens without global immunosuppression. Many immunomodulatory agents have been tested in both prevention and reversal of disease in preclinical studies and human clinical trials, yet report no durable efficacy. We developed a novel poly(lactic-co-glycolic) acid (PLGA) based microparticle (MP) formulation for the simultaneous delivery of multiple agents, tailored to induce a tolerogenic phenotype in dendritic cells (DC). This system employs large (～30uM diameter) MPs for sustained local controlled release, or small (～1 uM diameter) MPs that are readily engulfed by phagocytic cells. In studies performed in the non-obese diabetic (NOD) mouse, successful prevention of T1D was achieved with a quaternary MP therapy delivered subcutaneously: large MPs encapsulating TGF-β1 and GM-CSF, and small MPs encapsulating Vitamin D3 and denatured insulin. Features of this prevention included maturation-resistance in dendritic cells and the induction of regulatory T cell populations. Following these encouraging results, we began investigation of the effects of this formulation on human cells in vitro. Methods: Human monocyte-derived immature DCs (moDC) were generated from healthy subjects in the presence of GM-CSF and IL-4 for 7-9 days. Immature DCs were then incubated for 48 hours alone, with TGF-β1 MPs (1 mg/105 DCs) and Vitamin D3 MPs (10 MPs/DC), or with relevant unloaded MP controls at equivalent ratios. DCs were then treated with LPS (1 ug/ml) for 24 hours to assess maturation, or co-cultured with allogeneic dye-labeled T cells at a DC:T cell ratio of 1:4 to assess T cell proliferation in response to alloantigen. Results and Discussion: moDCs treated with factor-loaded MPs failed to upregulate HLA-DR and the costimulatory molecules CD40, CD80, and CD86 in response to LPS, indicative of maturation resistance(Fig. 1). Additionally, DCs exhibited increased expression of the negative regulators PD-L1 and Galectin 9(Fig. 2). Treated moDCs also suppressed alloantigen-induced T cell proliferation in both memory and naieve populations(Fig. 3). Conclusions: Characterization of human dendritic cell responses to this formulation in vitro is important to clinical translation. These results suggest similar mechanism of action in mouse preclinical studies and human cells in vitro, and provide support for clinical translation for immunotherapeutic application of this vaccine formulation.