Therapeutic Immunoregulation Mediated by TGF-beta-induced iTregs in Autoimmune Ar

描述（由申请人提供）： 目前的方法不能治愈类风湿性关节炎（RA）和其他慢性自身免疫性疾病。目前的提案计划开发一种新的方法来测试中心假设：CD 4？？用IL-2和TGF-2产生的调节性T细胞（iT细胞）在炎性环境中是稳定的，并且能够治疗胶原诱导的关节炎（CIA）。而胸腺衍生的，自然产生的CD 4？（nT细胞）抑制Th 1-或Th 2-细胞介导的自身免疫性疾病，这些细胞在控制（产生IL-17的）Th 17细胞介导的疾病如CIA中不太成功。此外，与iTclad不同，nTclad具有增加的细胞可塑性，并且可以转化为Th 1、Th 2或Th 17细胞，同时在促炎细胞因子的存在下失去其抑制活性。我们和其他人已经建立了TGF-2能够转换nove CD 4？细胞转化为iTcells，与nTcells具有相似的表型和功能特征。有趣的是，我们最近的研究表明，与nTclad不同，iTclad在促炎细胞因子的存在下不会转化为Th 17和Th 1细胞。此外，在体外IL-6的存在下，iTflu而不是nTflu维持对T细胞应答的抑制活性。此外，在IL-6和TGF-2的存在下，iT细胞而不是nT细胞甚至阻止其他T细胞变成Th 17细胞。然而，用IL- 2/TGF-2或atRA预处理的nT细胞改变了nT细胞的可塑性并恢复了功能。基于这些初步数据，我们预期iTdR和预处理的nTdR在过继转移到已建立的自身免疫性关节炎中后是稳定的。我们预期iTsalt和预处理的nTsalt可以显著改善治疗后已建立的关节炎的临床体征。我们还期望全反式维甲酸（atRA）和TGF-2或抗原特异性iTclad的组合可以增强iTclad对已建立的关节炎的治疗效果。我们认为，iT细胞不仅直接抑制T细胞应答，而且还诱导致耐受性DC的形成，并且这些DC产生IL-27、atRA和/或IDO，其最终抑制Th 17细胞分化和功能。因此，该项目有三个具体目标：1）确定当过继转移到建立的胶原诱导的关节炎（CIA）中时nTreg和iTreg细胞的相对稳定性。将从DBA/1 Foxp 3-GFP敲入小鼠或Foxp 3-GFP/IL-17-RFP双敲入小鼠中分选或诱导nTreg或iTreg。在用胶原II（CII）和完全弗氏佐剂（CFA）免疫后第14天或第28天，将nTreg或iTreg细胞过继转移到DBA/1或C57 BL/6小鼠中。将用GFP和RFP表达监测Treg亚群的迁移、分布、存活、表型（Foxp 3）和转化为辅助T细胞（Th 1、Th 2或Th 17细胞）。2)比较nTreg和iTreg细胞对胶原诱导性关节炎的治疗作用。在用CII/CFA免疫后第14天或第28天，将nTreg、iTreg或对照细胞施用至DBA/1 J小鼠。这些细胞的保护作用将通过关节炎发病率和严重程度、血清中抗CII IgG 2a抗体的水平和关节炎肢体的组织学检查来判断。3)定义iTclad抵抗Th 17细胞转化并调节Th 17细胞分化和炎症环境中功能的细胞和分子机制。我们将研究是否转录因子T-bet和Th 1细胞因子的表达在iT细胞负责他们的阻力。我们还将研究这些因子或由iTlR诱导的致耐受性DC是否有助于抑制Th 17分化和功能。本研究结果将促进对Treg细胞在类风湿关节炎防治中的作用的认识。如果成功，该项目也将具有直接的临床相关性，并可能提供一种治疗RA和其他自身免疫性疾病的新方法，这些疾病可能没有目前治疗的严重副作用。

DESCRIPTION (provided by applicant): Present approaches are unable to cure rheumatoid arthritis (RA) and other chronic autoimmune diseases. The current proposal plans to develop a novel approach that tests the central hypothesis: CD4?? regulatory T cells (iTregs) generated with IL-2 and TGF-2 are stable in an inflammatory milieu and are able to treat collagen-induced arthritis (CIA). While thymus- derived, naturally-occurring CD4???? (nTregs) suppress Th1- or Th2-cell-mediated autoimmune diseases, these cells are less successful in controlling (IL-17-producing) Th17 cell- mediated diseases such as CIA. Additionally, unlike iTregs, nTregs have increased cell plasticity, and can be converted into Th1, Th2 or Th17 cells while losing their suppressive activities in the presence of pro-inflammatory cytokines. We and others have established TGF-2 is able to convert naove CD4? cells to iTregs that share similar phenotypic and functional characteristics with nTregs. Interestingly, our recent studies revealed that unlike nTregs, iTregs did not make conversion to Th17 and Th1 cells in the presence of pro-inflammatory cytokines. In addition, iTregs but not nTregs maintained the suppressive activity against T cell response in the presence of IL-6 in vitro. Moreover, iTregs but not nTregs even prevented other T cells from becoming Th17 cells in the presence of IL-6 and TGF-2. However, pretreated nTregs with IL- 2/TGF-2 or atRA alters the plasticity and restore functionality of nTregs. Based on these preliminary data, we anticipate that iTregs and pretreated nTregs are stable following adoptive transfer into the established autoimmune arthritis. We expect iTregs and pretreated nTregs can significantly ameliorate the clinical signs of the established arthritis following treatment. We also expect that combination of all-trans retinoic acid (atRA) and TGF-2, or antigen-specific iTregs can enhance the therapeutic effects of iTregs on the established arthritis. We believe that iTregs not only directly suppress T cell response, but also induce the formation of tolerogenic DCs and these DCs produce IL-27, atRA and/or IDO that eventually restrain Th17 cell differentiation and function. Accordingly, the project has three specific aims: 1) Determine the relative stability of nTreg and iTreg cells when adoptively transferred into established collagen-induced arthritis (CIA). nTreg or iTregs will be sorted or induced from DBA/1 Foxp3-GFP knock-in mice or Foxp3-GFP/IL-17-RFP double knock-in mice. nTreg or iTregs cells will be adoptively transferred into DBA/1 or C57BL/6 mice at day 14 or day 28 after immunization with collagen II (CII) and Complete Freund's Adjuvant (CFA). The migration, distribution, survival, phenotype (Foxp3) and conversion into T help cells (Th1, Th2 or Th17 cells) of Treg subsets will be monitored with GFP and RFP expression. 2) Compare the therapeutic effect of both nTreg and iTreg cells on development of collagen-induced arthritis. nTreg, iTreg or control cells will be administrated to DBA/1J mice on day 14 or day 28 after immunization with CII/CFA. The protective effect of these cells will be judged by arthritis incidence and severity, levels of anti-CII IgG2a antibodies in sera and histological examination of arthritic limbs. 3) Define the cellular and molecular mechanism(s) by which iTregs are resistant to Th17 cell conversion and regulate Th17 cell differentiation and function in the inflammatory milieu. We will examine whether the transcription factor T-bet and Th1 cytokine expression in iTregs are responsible for their resistance. We will also examine whether these factors or tolerogenic DCs induced by iTregs contribute to suppressing Th17 differentiation and function. The results from this study will promote the understanding of therapeutic effects of Treg cells in the prevention and cure of rheumatoid arthritis. If successful, this project will also have a direct clinical relevance and possibly provide a novel approach to treat RA and other autoimmune diseases which may not have the severe side effect(s) characteristic of current therapies.