Tsuda Masato, Tone Yukiko, Ogawa Chihiro, Nagaoka Yoshiko, Katsumata Makoto, Necula Andra, Howie Duncan, Masuda Esteban, Waldmann Herman, Tone Masahide
Department of Biomedical Sciences, Cedars-Sinai Medical Center , Los Angeles, CA , USA.
Sir William Dunn School of Pathology, University of Oxford , Oxford , UK.
Front Immunol. 2017 Mar 13;8:279. doi: 10.3389/fimmu.2017.00279. eCollection 2017.
The transcription factor FOXP3 plays key roles in the development and function of regulatory T cells (Treg) capable of preventing and correcting immunopathology. There has been much interest in exploiting Treg as adoptive cell therapy in man, but issues of lack of nominal antigen-specificity and stability of FoxP3 expression in the face of pro-inflammatory cytokines have been a concern. In order to enable fundamental studies of human () gene regulation and to provide preclinical tools to guide the selection of drugs that might modulate hFOXP3 expression for therapeutic purposes, we generated hFOXP3/AmCyan bacterial artificial chromosome (BAC) transgenic mice and transfectants, wherein hFOXP3 expression was read out as AmCyan expression. Using the transgenic mice, one can now investigate gene expression under defined experimental conditions used for mouse Foxp3 (mFoxp3) studies. Here, we demonstrate that gene expression in BAC transgenic mice is solely restricted to CD4 T-cells, as for gene expression, showing that hFOXP3 expression in Treg cells depends on fundamentally similar processes to mFoxp3 expression in these cells. Similarly, hFOXP3 expression could be observed in mouse T-cells through TCR stimulation in the presence of TGF-β. These data suggest that, at least in part, cell type-specific human and mouse gene expression is regulated by common regulatory regions which for the human, are located within the 110-kb human FOXP3 BAC DNA. To investigate gene expression further and to screen potential therapeutics in modulating gene expression , we also generated hFOXP3/AmCyan expression reporter cell lines. Using the reporter cells and transcription factor inhibitors, we showed that, just as for mFoxp3 expression, inhibitors of NF-κB, AP1, STAT5, Smad3, and NFAT also block hFOXP3 expression. hFOXP3 induction in the reporter cells was also TGF-β dependent, and substantially enhanced by an mTOR inhibitor, Torin1. In both the reporter transgenic mice and cell lines, histone H4 molecules in the hFOXP3 promoter and enhancers located in human CNS1 and CNS2 regions were highly acetylated in natural Treg and TCR/TGF-β-induced Treg, indicating gene expression is regulated by mechanisms similar to those previously identified for the gene.
转录因子FOXP3在能够预防和纠正免疫病理的调节性T细胞（Treg）的发育和功能中发挥关键作用。人们对将Treg作为人类过继性细胞疗法有着浓厚兴趣，但面对促炎细胞因子时缺乏名义抗原特异性以及FoxP3表达稳定性的问题一直令人担忧。为了开展人类（）基因调控的基础研究，并提供临床前工具以指导选择可能用于调节hFOXP3表达以达到治疗目的的药物，我们构建了hFOXP3/AmCyan细菌人工染色体（BAC）转基因小鼠和转染细胞，其中hFOXP3表达通过AmCyan表达来读出。利用这些转基因小鼠，现在可以在用于小鼠Foxp3（mFoxp3）研究的特定实验条件下研究（）基因表达。在此，我们证明BAC转基因小鼠中的（）基因表达仅局限于CD4 T细胞，就如同（）基因表达一样，这表明Treg细胞中hFOXP3的表达依赖于与这些细胞中mFoxp3表达基本相似的过程。同样，在TGF-β存在的情况下，通过TCR刺激可在小鼠T细胞中观察到hFOXP3表达。这些数据表明，至少部分地，细胞类型特异性的人类和小鼠（）基因表达受共同调控区域调节，对于人类而言，这些区域位于110 kb的人类FOXP3 BAC DNA内。为了进一步研究（）基因表达并筛选调节（）基因表达的潜在治疗药物，我们还构建了hFOXP3/AmCyan表达报告细胞系。利用报告细胞和转录因子抑制剂，我们发现，正如mFoxp3表达一样，NF-κB、AP1、STAT5、Smad3和NFAT的抑制剂也会阻断hFOXP3表达。报告细胞中hFOXP3的诱导也依赖于TGF-β，并被mTOR抑制剂Torin1显著增强。在报告转基因小鼠和细胞系中，位于人类CNS1和CNS2区域的hFOXP3启动子和增强子中的组蛋白H4分子在天然Treg和TCR/TGF-β诱导的Treg中高度乙酰化，表明（）基因表达受与先前为（）基因所确定的机制相似的机制调节。
