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首页> 外文会议>World biomaterials congress >PLGA microparticle combination therapy induces a tolerogenic phenotype in human monocyte-derived dendritic cells
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PLGA microparticle combination therapy induces a tolerogenic phenotype in human monocyte-derived dendritic cells

机译：PLGA微粒联合疗法在人单核细胞衍生的树突状细胞中诱导耐受性表型

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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.
机译：简介：1型糖尿病（T1D）是一种自身免疫性疾病，其特征是对产生胰岛素的胰岛β细胞失去免疫耐受，然后被T细胞介导的破坏。一个主要的治疗目标是在没有整体免疫抑制的情况下，诱导持久的T细胞对β细胞抗原的耐受性。在临床前研究和人类临床试验中，已经在预防和逆转疾病中测试了许多免疫调节剂，但没有持久的疗效。我们开发了一种新颖的基于聚乳酸-乙醇酸（PLGA）的微粒（MP）制剂，用于同时递送多种药物，这些药物经定制可在树突状细胞（DC）中诱导耐受性表型。该系统采用大（〜30uM直径）MP进行局部局部控制释放，或采用小（〜1uM直径）MP吞噬细胞吞噬。在非肥胖型糖尿病（NOD）小鼠中进行的研究中，通过皮下给药的四级MP治疗成功地预防了T1D：包裹MP的大分子MP包裹TGF-β1和GM-CSF，包裹MP的小MP维生素D3和变性的胰岛素。这种预防的特征包括树突状细胞的抗成熟性和诱导调节性T细胞群体。取得这些令人鼓舞的结果后，我们开始研究这种制剂对人体细胞的体外作用。方法：在健康人存在GM-CSF和IL-4的情况下7-9天，从健康受试者中产生人单核细胞衍生的未成熟DC（moDC）。然后将未成熟的DC与TGF-β1MPs（1 mg / 105 DCs）和维生素D3 MPs（10 MPs / DC）或与相关的空载MP对照按等比例孵育48小时。然后用LPS（1 ug / ml）处理DC 24小时以评估其成熟度，或以1：4的DC：T细胞比率与同种异体染料标记的T细胞共培养以评估响应同种抗原的T细胞增殖。结果与讨论：载有因子的MP处理的moDC无法响应LPS来上调HLA-DR和共刺激分子CD40，CD80和CD86，表明其具有抗成熟性（图1）。此外，DC表现出负调节剂PD-L1和Galectin 9的表达增加（图2）。处理过的moDCs还抑制了同种异体抗原诱导的T细胞在记忆和幼稚群体中的增殖（图3）。结论：体外表征人树突状细胞对该制剂的反应对临床翻译很重要。这些结果表明在小鼠临床前研究和体外人细胞中有相似的作用机理，并为该疫苗制剂的免疫治疗应用的临床翻译提供支持。

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