Therapeutic immunoregulation mediated by TGF-beta-induced iTregs in autoimmune arthritis

摘要 目前最先进的类风湿性关节炎(RA)治疗方法并不能治愈，而且与 相当大的毒性。现在有确凿的证据表明，胸腺是自然产生的 CD4+CD25+Foxp3+调节性T细胞(NTreg)在预防自身免疫中的作用 包括类风湿性关节炎在内的疾病。数量减少与nTreg的功能障碍有关 与RA和其他自身免疫性疾病的发展和进展有关。虽然领养转让 NTreg对自身免疫性关节炎小鼠有明显的预防疾病进展的作用 这些细胞对已有疾病的治疗效果较差。主要原因是nTregs不稳定且 保留一种可塑性，使其能够转化为Th17/Th1效应细胞，从而降低其功能活性。 在我们之前的资助周期中，我们进行了两个里程碑式的观察：1)与nTreg不同，iTreg IL-2和转化生长因子-β体外诱导在有炎症的条件下稳定且有功能；2)nTreg 全反式维甲酸(AtRA)可以恢复它们的稳定性和治疗功能 在炎症和已确定的自身免疫性疾病中使用。鉴于发炎的人 滑膜组织和滑膜成纤维细胞是促炎细胞因子的主要来源，也是 自身免疫性关节炎的关节损害，我们将在此续订方案中测试iTreg是否保持其 炎症滑膜组织的表型和功能甚至抑制其增殖、迁移 炎症的滑膜细胞和组织产生细胞因子。我们提出了一组实验，使用 有条件的KO小鼠和人源化的动物模型将使我们能够理解其机制 由此iTreg诱导和nTreg修饰展示了它们在炎症反应中的稳定性和功能性 条件。我们预测DBC1，一个我们已经确定在这个过程中起关键作用的分子，是 在存在炎症的情况下对Treg的稳定性至关重要。我们还将研究关于以下方面的调查结果 稳定小鼠nTreg的方法可以推广到RA患者的人nTreg。至 为了实现这些目标，我们将制定以下四个具体目标的战略进行具体 实验。目的1：在滑膜组织发炎的情况下检查iTreg的命运。目标2： 确定iTreg和DC之间的串扰是否有助于iTreg对CIA的长期影响。目标3： 确定atRA是否维持RA患者nTreg的表型和功能稳定性，并目的 4.阐明DBC1在类风湿关节炎Treg中的体内外功能作用。什么时候 成功完成，这个项目将帮助我们了解Treg子集的特征 自身免疫性关节炎，可能导致RA和其他人类的潜在治疗策略 自身免疫性疾病。

Abstract Current state-of-the-art treatment of rheumatoid arthritis (RA) is not curative and is associated with considerable toxicity. Substantial evidence now exists that thymus-derived, naturally occurring CD4+CD25+Foxp3+ regulatory T cells (nTreg) play an important role in the prevention of autoimmune diseases including RA. Reduced numbers and the dysfunctional nature of nTreg have been associated with development and progression of RA and other autoimmune diseases. Although adoptive transfer of nTreg to mice with autoimmune arthritis can significantly prevent the disease progression, the effect of these cells on established disease is less effective. The major reason is that nTregs are unstable and retain a plasticity that allows them to convert to Th17/Th1 effector cells, diminishing their functional activity. In the cycle of our previous grant, we have made two milestone observations: 1) unlike nTreg, iTreg induced ex vivo with IL-2 and TGF-β are stable and functional in conditions with inflammation; 2) nTreg primed with all-trans retinoic acid (atRA) are able to restore their stability and functionality for therapeutic use in the presence of inflammation and in established autoimmune diseases. Given that the inflamed synovial tissue and synovial fibroblast are main sources of pro-inflammatory cytokines and key players for joint damage in autoimmune arthritis, we will test in this renewal proposal whether iTreg maintain their phenotype and function in the inflamed synovial tissues and even suppress the proliferation, migration and cytokine production of inflamed synovial cells and tissues. We propose a set of experiments using conditional KO mice and humanized animal models that will allow us to understand the mechanisms whereby iTreg induced and nTreg modified display their stability and functionality in the inflammatory condition. We predict that DBC1, a molecule that we have identified to have a key role in this process, is crucial for Treg stability in the presence of inflammation. We will also study whether the findings regarding the approach for stabilizing mouse nTreg can be extended to human nTreg from patients with RA. To achieve these aims, we will develop the strategies with the following four specific aims to conduct specific experiments. Aim 1: examine the fates of iTreg in the presence of inflamed synovial tissue. Aim 2: determine whether crosstalk between iTreg and DC contributes to long-term effects of iTreg on CIA. Aim 3: determine whether atRA maintains phenotypic and functional stability of nTreg from RA patients and Aim 4: elucidate the in vitro and in vivo functional role of DBC1 in Treg of rheumatoid arthritis. When successfully completed, this project will help us understand the characteristics of Treg subsets in autoimmune arthritis and may lead to a potential therapeutic strategy for RA and other human autoimmune diseases.