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J Immunol Methods. 2011 Sep 30;372(1-2):95-106. doi: 10.1016/j.jim.2011.07.001. Epub 2011 Jul 18.
Human regulatory T cells (Treg) are able to actively suppress autoreactive immune responses. Phenotypically, they are broadly characterized as CD4+, CD25+, CD127(lo/⁻) and FoxP3+. CD45RA can be used to further differentiate the population into naïve (CD45RA(+)) and induced (CD45RA⁻) Treg. The functional potential of Treg is routinely determined by assessing their ability to suppress T cell function in 3-5day proliferation assays. Since Treg are being explored for therapeutic use, a short-term functional assay could serve as a valuable tool for evaluating the potency of Treg. Therefore, an assay designed to measure Treg suppression of activation marker expression by responder T cells in 7 to 20h has been examined in this report. Using flow cytometry, expression of CD69 and CD154 on T cells, in response to stimulation with CD3/CD28 beads, was used as a measure of activation in the assay. Treg from healthy volunteers were sorted as CD4+CD25+CD127(lo/⁻)CD45RA+ cells with a BD FACSAria™ II. The highly purified Treg were then expanded in vitro and their function was assessed in short term activation marker suppression assays using autologous PBMC as responder cells. The data suggest that this short term suppression assay could be a reliable surrogate for assessing Treg functional potential.
人类调节性 T 细胞（Treg）能够主动抑制自身免疫反应。表型上，它们广泛表现为 CD4+、CD25+、CD127(lo/-)和 FoxP3+。CD45RA 可用于进一步将该群体分为幼稚（CD45RA(+)）和诱导（CD45RA⁻）Treg。Treg 的功能潜力通常通过评估其在 3-5 天增殖测定中抑制 T 细胞功能的能力来确定。由于 Treg 正在被探索用于治疗用途，短期功能测定可以作为评估 Treg 效力的有价值工具。因此，本报告中检查了一种旨在测量 Treg 在 7 至 20 小时内抑制应答 T 细胞激活标志物表达的测定方法。通过流式细胞术，使用 CD3/CD28 珠刺激后 T 细胞上 CD69 和 CD154 的表达，作为测定中激活的衡量标准。健康志愿者的 Treg 通过 BD FACSAria™ II 作为 CD4+CD25+CD127(lo/-)CD45RA+细胞进行分选。然后，将高度纯化的 Treg 在体外扩增，并使用自体 PBMC 作为应答细胞在短期激活标志物抑制测定中评估其功能。数据表明，这种短期抑制测定可能是评估 Treg 功能潜力的可靠替代方法。
