BACKGROUND Several methods exist for flow-cytometric estimation of human peripheral blood CD4+ T regulatory cells (CD4+ Tregs). METHODS We report our experience with the estimation of human CD4+ Tregs via three different characterizations using flow cytometry (CD25high FoxP3+ , CD25high CD127low/- FoxP3+ , and CD4+ CD25high/int CD45ROFoxP3+ ) in normal subjects. We have used these methods on the control populations from two studies (32 and 36 subjects, respectively), the latter two methods retrospectively on the subjects of the first study. The six CD4+ T cell fractions obtained by the third method were differentially colored to ascertain the distribution of these cell fractions in the CD25/FoxP3, CD45RO/FoxP3, and CD25/CD127 dot plots from CD4/CD25/CD45RO/FoxP3 and CD4/CD25/CD45RO/CD127 panels. RESULTS Each approach gives significantly different estimates of Tregs (expressed as percentage of CD4+ T cells), with the second almost invariably yielding higher percentages than the other two. Only the third approach can distinguish among effector and naïve Tregs and FoxP3+ non-Tregs. Analysis of CD25/CD127 dot plots reveals that Treg delineation via the widely used definition of CD4+ CD25high CD127low/- cells unavoidably yields a mixture of nearly all effector and most of naïve Tregs, as well as FoxP3+ non-Tregs plus other cells. Delineation of effector/naïve Tregs and FoxP3+ non-Tregs is possible via CD45RO/CD25 dot plots but not by CD45RO/FoxP3 counterparts (as done previously) because of overlapping FoxP3 intensities among Tregs and non-Tregs. CONCLUSION Our comparison shows that CD4/CD25/CD45RO/FoxP3 panels are an objective means of estimating effector and naïve Tregs via colored dot plots, aiding thus in Treg delineation in health and detecting aberrations in disease.

中文翻译：

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一种改进的流式细胞术方法，用于通过CD4 / CD25 / CD45RO / FoxP3标记客观估计外周血中的人CD4 +调节性T细胞（CD4 + Treg）。

背景技术存在几种用于流式细胞术估计人外周血CD4 + T调节细胞（CD4 + Treg）的方法。方法我们报告了我们在正常受试者中通过流式细胞术（CD25high FoxP3 +，CD25high CD127low /-FoxP3 +和CD4 + CD25high / int CD45ROFoxP3 +）通过三种不同的表征估算人类CD4 + Treg的经验。我们已经在两项研究（分别为32和36个受试者）的对照人群中使用了这些方法，而后两种方法则是对第一个研究的受试者进行回顾性研究。对通过第三种方法获得的六个CD4 + T细胞级分进行差异着色，以确定这些细胞级分在CD4 / CD25 / CD45RO / FoxP3和CD4 / CD25的CD25 / FoxP3，CD45RO / FoxP3和CD25 / CD127点图中的分布/ CD45RO / CD127面板。结果每种方法对Treg的估计值（以CD4 + T细胞的百分比表示）有显着不同，第二种方法几乎总是比其他两种方法产生更高的百分比。只有第三种方法可以区分效应子和幼稚Treg和FoxP3 +非Treg。CD25 / CD127点图的分析显示，通过广泛使用的CD4 + CD25high CD127low /-细胞定义，Treg轮廓不可避免地产生了几乎所有效应子和大多数Treg以及FoxP3 +非Treg以及其他细胞的混合物。由于Treg和非Treg之间的FoxP3强度重叠，因此可以通过CD45RO / CD25点图来描绘效应子/纯真Treg和FoxP3 +非Treg，但不能通过CD45RO / FoxP3对应物（如前所述）来描绘。