OMIP-015: Human regulatory and activated T-cells without intracellular staining
OMIP-015: Human regulatory and activated T-cells without intracellular staining
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DOI：
10.1002/cyto.a.22230
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发表时间：
2013-02-01
期刊：
影响因子：
3.7
通讯作者：
Roederer, Mario
中科院分区：
文献类型：
--
作者：
Mahnke, Yolanda D.;Beddall, Margaret H.;Roederer, Mario
研究基础最近发表了两种用于研究Treg的OMIP（1，2）。这些使用表面CD 25和细胞内FoxP 3的表达来鉴定CD 4 + T细胞的Treg亚群。该面板旨在避免由固定、透化和细胞内染色引起的实验复杂性。值得注意的是，该小组可能与Treg的功能和基因组询问所需的活细胞分选相容。使用IL-2 Ra链（CD 25）和IL-7 Ra链（CD 127）的组合测量，其中Treg定义为CD 25 hi CD 127 J（3）。只有大约13%的人是FoxP 3 J（未发表的数据）（3，4）。通过CD 39、CD 73、CD 45 RA、CD 45 RO和PD-1的表达进一步表征Treg的功能和表型。三磷酸核苷去磷酸化酶CD 39已被证明可以识别具有体外免疫抑制能力的细胞（5，6）。CD 73是一种胞外-50-核苷酸酶，与CD 39一起通过产生腺苷促进Treg和其他抑制性免疫细胞以及肿瘤微环境的抑制功能（7-9）。包括CD 45 RA（10）和CD 45 RO（11）以分别定义初始和活化的Treg，而选择PD-1，因为它已被证明负调节Treg功能（12）。虽然本小组的主要目的是鉴定和表征Treg，但所包括的一些标志物还提供了关于其他CD 4+和CD 8 + T细胞的分化和活化状态的信息。例如，尽管CD 39主要在免疫抑制性Treg上表达，但也在致病性CD 4 + CD 25 J效应记忆T细胞上发现（13）。CD 45 RA、CD 45 RO和CD 127在T细胞分化期间差异表达，而CD 25和PD-1以活化依赖性方式表达。最后，包括CD 38和HLA-DR;这两种T细胞活化标志物的差异表达鉴定了与疾病控制相关的亚群，特别是在HIV-1感染中（14，15）。为了探索Treg上活化标志物的表达，在总CD 4 + T细胞上设置门（图1B），然后应用于Treg（图1C）。对于暗淡的标志物如HLA-DR和PD-1，仔细选择在点图中可视化的第二标志物允许鉴定用于分离阳性和阴性的最佳阈值。
BACKGROUNDTwo OMIPs designed for the investigation of Treg have recently been published (1, 2). These use the expression of surface CD25 and intracellular FoxP3 to identify the Treg subset of CD4+ T-cells. This panel is designed to avoid experimental complexity induced by fixation, permeabilization, and intracellular staining. Notably this panel is potentially compatible with live cell sorting needed for functional and genomic interrogation of Treg. A combined measurement of the IL-2Ra chain (CD25) and the IL-7R a-chain (CD127) was used, with Treg being defined as CD25hi CD127J (3). Only about 13% of this population is FoxP3J (unpublished data)(3, 4). Treg were further characterized functionally and phenotypically by the expression of CD39, CD73, CD45RA, CD45RO, and PD-1. The nucleoside triphosphate dephosphorylase CD39 has been shown to identify cells with in vitro immunosuppressive capacity (5, 6). CD73 is an ecto-50-nucleotidase that, together with CD39, contributes to the inhibitory function of Treg and other suppressive immune cells, as well as the tumor microenvironment, by generating adenosine (7–9). CD45RA (10) and CD45RO (11) were included in order to define naıve and activated Treg, respectively, while PD-1 was selected as it has been demonstrated to negatively regulate Treg function (12). While the primary objective of the present panel was to identify and characterize Treg, some of the markers included also provide information on the differentiation and activation status of other CD4+ and CD8+ T-cells. For instance, though CD39 is mainly expressed on immunosuppressive Treg, it is also found on pathogenic CD4+ CD25J effector memory T-cells (13). CD45RA, CD45RO, and CD127 are differentially expressed during T-cell differentiation, while CD25 and PD-1 are expressed in an activation-dependent manner.Finally, CD38 and HLA-DR were included; differential expression of these two T-cell activation markers identifies subsets disparately correlating with disease control, particularly in HIV-1 infection (14, 15). To explore the expression of activation markers on Treg, gates were set on total CD4+ T-cells (Fig. 1B) and then applied to Treg (Fig. 1C). For dim markers such as HLA-DR and PD-1, careful choice of the second marker visualized in the dot plot allowed identifying the best threshold for separating positive and