Therapeutic immunoregulation mediated by TGF-??-induced iTregs in autoimmune arth

描述(由申请人提供)： 目前的治疗方法无法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说：CD4？？由IL-2和TGF-2产生的调节性T细胞(ITregs)在炎症环境中稳定，能够治疗胶原性关节炎(CIA)。而胸腺来源的自然产生的CD4？(NTregs)抑制Th1或Th2细胞介导的自身免疫性疾病，这些细胞在控制(IL-17产生)Th17细胞介导的疾病(如CIA)方面不太成功。此外，与iTregs不同，nTregs具有增强的细胞可塑性，可以转化为Th1、Th2或Th17细胞，同时在促炎细胞因子存在的情况下失去抑制活性。我们和其他人已经建立了转化生长因子-2是否能够转化为NAOVE CD4？ITregs的表型和功能特征与nTregs相似。有趣的是，我们最近的研究表明，与nTregs不同，iTregs在促炎细胞因子存在的情况下不会转化为Th17和Th1细胞。此外，iTregs而不是nTregs在体外保持了在IL-6存在下对T细胞反应的抑制活性。此外，iTregs而不是nTregs甚至在IL-6和TGF-2存在的情况下阻止其他T细胞成为Th17细胞。然而，经IL-2/TGF-2或atRA处理的nTregs改变了nTregs的可塑性和恢复功能。基于这些初步数据，我们预计iTregs和预处理的nTregs在过继转移到已建立的自身免疫性关节炎后是稳定的。我们预计iTregs和经过预处理的nTregs可以在治疗后显著改善已建立的关节炎的临床症状。我们还期待全反式维甲酸(AtRA)和转化生长因子-2或抗原特异性iTregs的结合可以增强iTregs对已建立的关节炎的治疗效果。我们认为iTregs不仅直接抑制T细胞的反应，还能诱导产生耐受性DC，这些DC产生IL-27、atRA和/或IDO，最终抑制Th17细胞的分化和功能。因此，该项目有三个具体目标：1)确定过继转移到已建立的胶原性关节炎(CIA)中时nTreg和iTreg细胞的相对稳定性。从DBA/1 Foxp3-GFP敲入小鼠或Foxp3-GFP/IL-17-RFP双重敲入小鼠中分离或诱导出nTreg或iTregs。在II型胶原(CII)和完全弗氏佐剂(CFA)免疫后第14天或第28天，将nTreg或iTregs细胞过继转移到DBA/1或C57BL/6小鼠体内。GFP和RFP的表达将监测Treg亚群的迁移、分布、存活、表型(Foxp3)和向T辅助细胞(Th1、Th2或Th17细胞)的转化。2)比较nTreg和iTreg细胞对胶原性关节炎的治疗作用。在CII/CFA免疫后第14天或第28天分别给DBA/1J小鼠注射nTreg、iTreg或对照细胞。这些细胞的保护作用将通过关节炎的发生率和严重程度、血清中抗CII IgG2a抗体水平和四肢关节炎的组织学检查来判断。3)明确iTregs抵抗Th17型细胞转化的细胞和分子机制(S)，调节炎症环境中Th17型细胞的分化和功能。我们将研究转录因子T-bet和Th1细胞因子在iTregs中的表达是否与它们的耐药性有关。我们还将研究这些因素或iTregs诱导的耐受性DC是否有助于抑制Th17的分化和功能。这项研究的结果将促进对Treg细胞在防治类风湿性关节炎中的治疗作用的理解。如果成功，该项目还将具有直接的临床意义，并可能提供一种新的方法来治疗类风湿性关节炎和其他可能没有现有疗法所特有的严重副作用(S)的自身免疫性疾病。 公共卫生相关性： 拟议的研究将确定CD_4、CD_4、CD_4IL-2和转化生长因子-2产生的细胞可治疗胶原诱导的自身免疫性类风湿性关节炎。因此，这一研究项目的成果不仅将有助于我们了解转化生长因子-2诱导的调节性T细胞在关节炎抗炎活性中的生物学作用，而且将为人类类风湿关节炎的潜在治疗和预防提供一种新的有效的细胞疗法。考虑到超过1%的美国人患有类风湿性关节炎，而目前的治疗方法无法治愈这种疾病，这将产生巨大的科学和经济影响。

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. PUBLIC HEALTH RELEVANCE: The proposed research will determine whether the CD4?? cells generated with IL-2 and TGF-2 can treat autoimmune rheumatoid arthritis in collagen-induced arthritis model. Thus, accomplishments from this proposed research project will not only help us understand the biological role of TGF-2-induced regulatory T cells in the anti-inflammatory activity in arthritis, but also lead to a novel and effective cell therapy for potential treatment and prevention in human rheumatoid arthritis. This will have an enormous scientific and economic impact, considering the fact that more than 1% of Americans are suffering from rheumatoid arthritis and current therapeutic approaches are unable to cure this disease.