Schmidt Angelika, Éliás Szabolcs, Joshi Rubin N, Tegnér Jesper
Unit of Computational Medicine, Center for Molecular Medicine, Department of Medicine Solna, Karolinska Institutet, Karolinska University Hospital, & Science for Life Laboratory;
Unit of Computational Medicine, Center for Molecular Medicine, Department of Medicine Solna, Karolinska Institutet, Karolinska University Hospital, & Science for Life Laboratory.
J Vis Exp. 2016 Dec 30(118):55015. doi: 10.3791/55015.
Regulatory T cells (Tregs) are an integral part of peripheral tolerance, suppressing immune reactions against self-structures and thus preventing autoimmune diseases. Clinical approaches to adoptively transfer Tregs, or to deplete Tregs in cancer, are underway with promising first outcomes. Because the number of naturally occurring Tregs (nTregs) is very limited, studying certain Treg features using in vitro induced Tregs (iTregs) can be advantageous. To date, the best although not absolutely specific protein marker to delineate Tregs is the transcription factor FOXP3. Despite the importance of Tregs including non-redundant roles of peripherally induced Tregs, the protocols to generate iTregs are currently controversial, particularly for human cells. This protocol therefore describes the in vitro differentiation of human CD4+FOXP3+ iTregs from human naïve T cells using a range of Treg-inducing factors (TGF-β plus IL-2 only, or their combination with retinoic acid, rapamycin or butyrate) in parallel. It also describes the phenotyping of these cells by flow cytometry and qRT-PCR. These protocols result in reproducible expression of FOXP3 and other Treg signature genes and enable the study of general FOXP3-regulatory mechanisms as well as protocol-specific effects to delineate the impact of certain factors. iTregs can be utilized to study various phenotypic aspects as well as molecular mechanisms of Treg induction. Detailed molecular studies are facilitated by relatively large cell numbers that can be obtained. A limitation for the application of iTregs is the relative instability of FOXP3 expression in these cells compared to nTregs. iTregs generated by these protocols can also be used for functional assays such as studying their suppressive function, in which iTregs induced by TGF-β plus retinoic acid and rapamycin display superior suppressive activity. However, the suppressive capacity of iTregs can differ from nTregs and the use of appropriate controls is crucial.
调节性T细胞（Tregs）是外周耐受的重要组成部分，可抑制针对自身结构的免疫反应，从而预防自身免疫性疾病。过继性转移Tregs或在癌症中清除Tregs的临床方法正在进行中，并取得了令人鼓舞的初步成果。由于天然存在的Tregs（nTregs）数量非常有限，因此使用体外诱导的Tregs（iTregs）研究某些Treg特征可能具有优势。迄今为止，用于描绘Tregs的最佳（尽管不是绝对特异性的）蛋白质标志物是转录因子FOXP3。尽管Tregs很重要，包括外周诱导的Tregs的非冗余作用，但目前生成iTregs的方案仍存在争议，尤其是对于人类细胞。因此，本方案描述了使用一系列Treg诱导因子（仅TGF-β加IL-2，或它们与视黄酸、雷帕霉素或丁酸盐的组合）从人幼稚T细胞体外分化出人CD4 + FOXP3 + iTregs的过程。它还描述了通过流式细胞术和qRT-PCR对这些细胞进行表型分析的方法。这些方案可导致FOXP3和其他Treg特征基因的可重复表达，并有助于研究一般的FOXP3调节机制以及特定方案的效应，以描绘某些因素的影响。iTregs可用于研究Treg诱导的各种表型方面以及分子机制。通过可获得的相对大量细胞便于进行详细的分子研究。iTregs应用的一个限制是与nTregs相比，这些细胞中FOXP3表达相对不稳定。通过这些方案产生的iTregs也可用于功能测定，例如研究它们的抑制功能，其中由TGF-β加视黄酸和雷帕霉素诱导的iTregs表现出卓越的抑制活性。然而，iTregs的抑制能力可能与nTregs不同，使用适当的对照至关重要。
