Husseiny Mohamed I, Fahmy Ahmed, Du Weiting, Gu Angel, Garcia Pablo, Ferreri Kevin, Kandeel Fouad
Department of Translational Research & Cellular Therapeutics, Diabetes & Metabolism Research Institute, Beckman Research Institute of City of Hope, Duarte, CA, United States.
Faculty of Pharmacy, Zagazig University, Zagazig, Egypt.
Front Genet. 2020 Apr 7;11:300. doi: 10.3389/fgene.2020.00300. eCollection 2020.
Regulatory T cells (Tregs) suppress immune responses in an antigen-specific manner. Of clinical relevance, Tregs can be isolated and expanded while maintaining immunoregulatory function. Tregs are classified as CD4CD25CD127 FOXP3 cells. Demethylation of the Treg-specific demethylation region (TSDR) of FOXP3 is found in natural Tregs (nTregs). We report a method for the characterization of the differential methylation pattern of the FOXP3 TSDR in patient-derived and expanded nTregs. Human TSDR sequences from nTregs (unmethylated sequence) and pancreatic (methylated sequence) cells were amplified and cloned into plasmids. A droplet digital TaqMan probe-based qPCR (ddPCR) assay using methylation-specific primers and probes was employed to quantify unmethylated and methylated sequences. The methylation-specific droplet digital PCR (ddMSP) assay was specific and selective for unmethylated DNA in mixtures with methylated DNA in the range of 5000 copies/μL to less than 1 copy/μL ( = 0.99) even in the presence of non-selective gDNAs. CD4CD25CD127FOXP3 human nTregs, in the presence of Dynabeads or activators, were expanded for 21 days. There was a decrease in the unmethylated ratio of Tregs after expansion with essentially the same ratio at days 10, 14, and 17. However, the activator expanded group showed a significant decrease in unmethylated targets at day 21. The suppression activity of activator-expanded nTregs at day 21 was decreased compared to cells expanded with Dynabeads. These data suggest that the ddMSP can quantitatively monitor nTreg expansion . These data also indicate that the assay is sensitive and specific at differentiating nTregs from other cells and may be useful for rapid screening of nTregs in clinical protocols.
调节性T细胞（Tregs）以抗原特异性方式抑制免疫反应。具有临床相关性的是，Tregs可以在保持免疫调节功能的同时进行分离和扩增。Tregs被分类为CD4CD25CD127 FOXP3细胞。在天然Tregs（nTregs）中发现了FOXP3的Treg特异性去甲基化区域（TSDR）的去甲基化。我们报告了一种用于表征患者来源并扩增的nTregs中FOXP3 TSDR差异甲基化模式的方法。来自nTregs（未甲基化序列）和胰腺（甲基化序列）细胞的人类TSDR序列被扩增并克隆到质粒中。使用甲基化特异性引物和探针的基于液滴数字TaqMan探针的qPCR（ddPCR）测定法用于定量未甲基化和甲基化序列。甲基化特异性液滴数字PCR（ddMSP）测定法对于含有5000拷贝/μL至小于1拷贝/μL范围内甲基化DNA的混合物中的未甲基化DNA具有特异性和选择性（r = 0.99），即使存在非选择性基因组DNA也是如此。在存在磁珠或激活剂的情况下，将CD4CD25CD127FOXP3人类nTregs扩增21天。扩增后，Tregs的未甲基化比例降低，在第10、14和17天基本相同。然而，激活剂扩增组在第21天未甲基化靶点显著降低。与用磁珠扩增的细胞相比，激活剂扩增的nTregs在第21天的抑制活性降低。这些数据表明ddMSP可以定量监测nTreg扩增。这些数据还表明该测定法在区分nTregs与其他细胞方面具有敏感性和特异性，可能有助于临床方案中nTregs的快速筛选。
