Abstract
BackgroundMultiple 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 FindingsTregs 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/SignificanceWe 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.
Highlights
Numerous factors have been reported to account for CD4+FOXP3+ regulatory T cell (Treg)-mediated immune suppression including those that are cytotoxic such as perforin and granzyme B [1] and those that suppress proliferation and cytokine secretion in target cells
The possibility that Tregs directly induce oxidant stress in T effector cells (Teffs) has not widely investigated as a mechanism of suppression. In light of these previous findings, we evaluated the role of reactive oxygen species (ROS) in direct Treg-mediated suppression of Teffs using antioxidants, NADPH oxidase inhibitors and T cells from Ncf1-deficient (Ncf12/2) mice that lack a functional NADPH oxidase complex due to a substitution mutation that results in aberrant Ncf1 gene splicing
In order to assess the potential contribution of ROS to Treg suppression of Teffs we performed conventional Treg suppression assays, using wild type cells and a 1:1 ratio of Tregs to Teffs, in the presence of the antioxidant NAC to block ROS
Summary
Numerous factors have been reported to account for CD4+FOXP3+ regulatory T cell (Treg)-mediated immune suppression including those that are cytotoxic such as perforin and granzyme B [1] and those that suppress proliferation and cytokine secretion in target cells The latter category includes factors such as IL-10, TGFb and CTLA-4 and mechanisms such as suppression of IL-2 production by target cells or Il-2 sequestration by Tregs [2]. The existing literature indicates that this occurs by both direct mechanisms, which can be measured in in vitro co-culture assays, and by indirect mechanisms where Tregs modulate the activity of dendritic cells and macrophages that, in turn, affect suppression. The latter scenarios are likely best evaluated in in vivo models. Given the inherent redox sensitivity of T cells, we tested the hypothesis that oxidants may mediate the direct suppression of Teffs by Tregs
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