Department of Pathology, University of Utah, Salt Lake City, Utah, United States of America.
PLoS One. 2011 Jan 6;6(1):e16013. doi: 10.1371/journal.pone.0016013.
Multiple mechanisms have been advanced to account for CD4+FOXP3+ regulatory T cell (Treg)-mediated suppression of CD4+ effector T cells (Teffs) but none appear to completely explain suppression. Previous data indicates that Tregs may affect the microenvironment redox state. Given the inherent redox sensitivity of T cells, we tested the hypothesis that oxidants may mediate the direct suppression of Teffs by Tregs.
METHODOLOGY/PRINCIPAL FINDINGS: Tregs and Teffs were isolated from the spleens of wild type (WT) C57BL/6 mice or Ncf1(p47phox)-deficient C57BL/6 mice which lack NADPH oxidase function. Teffs were labeled with CFSE and co-cultured with unlabeled Tregs at varying Treg:Teff ratios in the presence of anti-CD3/CD28 coated beads for 3 days in suppression assays. Treg-mediated suppression was quantified by flow cytometric analysis of CFSE dilution in Teffs. The presence of the antioxidants n-acetylcysteine (NAC) or 2-mercaptoethanol or inhibitors of NADPH oxidase (diphenyleneiodonium and VAS-2870) resulted in reduced WT Treg-mediated suppression. The observed suppression was in part dependent upon TGFβ as it was partially blocked with neutralizing antibodies. The suppression of Teff proliferation induced by exogenous TGFβ treatment could be overcome with NAC. Ncf1-deficient Teff were slightly but significantly less sensitive than WT Teff to suppression by exogenous TGFβ. Ncf1-deficient Tregs suppressed Ncf1-deficient Teff very poorly compared to wild type controls. There was partial but incomplete reconstitution of suppression in assays with WT Tregs and Ncf1-deficient Teff.
CONCLUSIONS/SIGNIFICANCE: We present evidence that NADPH oxidase derived ROS plays a role in the direct Treg mediated suppression of CD4+ effector T cells in a process that is blocked by thiol-containing antioxidants, NADPH oxidase inhibitors or a lack of Ncf1 expression in Tregs and Teffs. Oxidants may represent a potential new target for therapeutic modulation of Treg function.
已经提出了多种机制来解释 CD4+FOXP3+调节性 T 细胞（Treg）介导的 CD4+效应 T 细胞（Teffs）抑制，但没有一种机制似乎能完全解释这种抑制。先前的数据表明，Tregs 可能会影响微环境的氧化还原状态。鉴于 T 细胞固有的氧化还原敏感性，我们检验了这样一种假设，即氧化剂可能介导 Tregs 对 Teffs 的直接抑制。
方法/主要发现：从野生型（WT）C57BL/6 小鼠或缺乏 NADPH 氧化酶功能的 Ncf1（p47phox）缺陷型 C57BL/6 小鼠的脾脏中分离出 Tregs 和 Teffs。Teffs 用 CFSE 标记，并在存在抗 CD3/CD28 包被珠的情况下，与未标记的 Tregs 以不同的 Treg:Teff 比例共培养 3 天，进行抑制实验。通过流式细胞术分析 Teffs 中 CFSE 稀释来定量 Treg 介导的抑制。抗氧化剂 N-乙酰半胱氨酸（NAC）或 2-巯基乙醇或 NADPH 氧化酶抑制剂（二苯并碘onium 和 VAS-2870）的存在导致 WT Treg 介导的抑制减少。观察到的抑制部分依赖于 TGFβ，因为它可以被中和抗体部分阻断。用外源性 TGFβ 处理诱导的 Teff 增殖抑制可以被 NAC 克服。与 WT Teff 相比，Ncf1 缺陷型 Teff 对 TGFβ 抑制的敏感性略低，但差异显著。与野生型对照相比，Ncf1 缺陷型 Tregs 对 Ncf1 缺陷型 Teff 的抑制作用非常差。在用 WT Tregs 和 Ncf1 缺陷型 Teff 进行的实验中，抑制作用部分但不完全重建。
结论/意义：我们提供的证据表明，NADPH 氧化酶衍生的 ROS 在 Treg 介导的 CD4+效应 T 细胞的直接抑制中发挥作用，该过程被含巯基的抗氧化剂、NADPH 氧化酶抑制剂或 Tregs 和 Teffs 中 Ncf1 表达的缺乏所阻断。氧化剂可能是调节 Treg 功能的一个潜在的新靶点。
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