Autoreactive T cells that are capable of inducing disease exist in normal adult animals, but are maintained in a dormant or inactive state due to the suppressive functions of CD4+CD25+Foxp3+ regulatory T cells (Treg). Our recent studies have focused on defining the mechanism of action of the CD4+CD25+Foxp3+ Treg cells in vitro and on an analysis of their potential dysfunction in autoimmune disease: (1) We are analyzing the role of the transcription factor Helios (Ikzf2) in Treg function. Helios is a transcription factor belonging to the zinc finger containing Ikaros family comprised of five transcription factors-- Ikaros, Helios, Aiolos, Eos and Pegasus. Ikaros is expressed in all immune cells. Ikaros has the capacity to dimerize, not only with other Ikaros isoforms, but also with Aiolos or Helios isoforms. Ikaros can act both as a repressor and an activator of gene transcription. Helios can dimerize with itself, as well as with other family members. The Helios gene (Ikzf2) encodes protein isoforms with zinc-finger domains exhibiting considerable homology to Ikaros family proteins. Human and mouse Helios share 97% identity in their amino acid sequences. We screened a differentially expressed cDNA library from CD4+CD25+ T cells and found that helios was expressed at much higher levels when compared to CD4+CD25- T cells. Real-time PCR confirmed that helios mRNA is expressed at a rate 75-fold greater in CD4+CD25+ T cells than in CD4+CD25- T cells. To further characterize Helios, we examined Helios mRNA expression in various thymic subpopulations. We observed that Helios was expressed in the thymus at low levels as early as the DN stage. Helios continued to be expressed at low levels in DP cells, CD8+ cells and CD4+CD25- cells. However, there was a striking increase in Helios mRNA in thymic CD4+CD25+ cells, concurrent with the first appearance of Foxp3 mRNA. In order to more thoroughly examine Helios expression and function, we generated a monoclonal antibody (22F6) to a large peptide within the non-conserved 110 amino acid region in Helios. In agreement with the qPCR, mAb 22F6 reacted preferentially with CD4+CD25+ Tregs. Notably, when LN cells were gated on CD4+ cells and analyzed for Foxp3 and Helios expression, Helios was expressed on only 70% of Foxp3+ cells. As 22F6 was raised to a mouse peptide that differed from the human sequence by only two amino acids, we analyzed the reactivity of 22F6 in human cells. 22F6 also reacted with human PBMCs and was again, specific to the CD4+CD25hi Treg subset. Although much more variable than in mouse CD4+ cells, Helios was expressed in a subset of CD4+Foxp3+ cells in human that ranged from 50-90%, but on average, comprised 70% of Foxp3+ cells. Helios was expressed at the DN stage of thymic development, in agreement with the quantitative PCR data. Specifically, Helios was expressed at low levels as early as DN1, but was expressed in 100% of DN2 and DN3 cells. By DN4, Helios expression had begun to decrease. In contrast, Foxp3 was not expressed at the DN stage and thus, Helios precedes Foxp3 expression. We also observed that percentage of CD4+Foxp3+ Helios- cells was significantly lower in the thymus than in the periphery. Notably, there are no Foxp3+ Helios- cells at day 3 of life. Foxp3+ Helios- cells are absent in the neonate, do not significantly appear in the spleen until between day 7 and day 14 of life and do not reach the percentage observed in an adult until after the pups have been weaned. The observation that Foxp3+Helios- cells appear to arise in peripheral lymphoid tissue and do not acquire their full phenotypic profile until after weaning led us to examine the expression of Helios in TGF-beta induced iTreg cells. Both human and mouse iTreg were helios-. Collectively, these results demonstrate that Helios is a specific marker of thymic-derived Treg cells and raise the possibility that a significant percentage of Foxp3+ Treg cells are generated extra-thymically. (2)Statins are widely used drugs for the treatment of hypercholesterolemia. They function as competitive inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A reductase, which is the rate-determining enzyme of the mevalonate pathway. More recent studies have also suggested that statins can mediate immunosuppressive functions and have proven effective in the treatment of autoimmune diseases or graft-versus-host disease in animal models. Although direct effects of statins on Treg function have not been reported, a number of studies have suggested that Treg play an important role in the control of pathology in atherosclerosis and atherosclerotic plaques have been reported to contain a lower percentage of Foxp3+ Tregs compared to normal tissue. We have evaluated the effects of simvastatin blockade of the mevalonate pathway on the induction of Foxp3 expressing iTregs in vitro. We demonstrate that simvastatin itself can mediate induction of Foxp3+ T cells and can also synergize with low levels of TGF-beta in the induction of functional Foxp3+ Treg. The effects of simvastatin are secondary to a blockade of protein geranylgeranylation, are mediated 24 hours after TCR stimulation, and are associated with TCR-specific DNA demethylation of the Foxp3 promoter and TCR-specific induction of Smad6 and 7 proteins. Although it is difficult to extrapolate from our in vitro models systems to the in vivo situation, our results that simvastatin can markedly enhance the induction of Foxp3 expression in the presence of low concentrations of TGF-beta strongly suggest that some of the beneficial effects of simvastatin include the generation of Tregs in the inflammatory milieu of the atherosclerotic plaque.
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