Department of Hematology and Transplantology, Medical University of Gdansk, 80-214 Gdansk, Poland.
Cancer Research Center of Toulouse, Inserm UMR 1037, CNRS ERL 5294, University of Toulouse, Oncopole, Toulouse, France.
Folia Histochem Cytobiol. 2021;59(2):75-85. doi: 10.5603/FHC.a2021.0014. Epub 2021 Jun 7.
Regulatory T cells (Tregs) are a unique CD4+ T cell subset involved in the regulation of immune responses. The traditional immunophenotype used to define Tregs includes CD4+CD25high and the expression of the transcription factor Forkhead box protein 3 (FoxP3). A complex technique of intracellular staining, transient upregulation of FoxP3 in activated conventional T lymphocytes (Tcons), and the omission of naïve CD45RA+ Tregs with downregulated FoxP3 activity but a demethylated FOXP3 promoter region may lead to inaccurate quantification. In an attempt to meet the need for a reliable and simplified enumeration strategy, we investigated different membrane markers to capture the entire Treg compartment and to identify subpopulations of Tregs.
Analyses were performed on whole blood. Tested gating strategies were based on the expression of the following membrane antigens: CD45, CD3, CD4, CD25, CD127, CD26, CD6, CD39, CD71, HLA-DR, CD45RA and CD31. Double controls with FoxP3 were performed.
The final enumeration panel consisted of the membrane markers CD45, CD3, CD4, CD25, CD127, CD26, CD39, CD45RA and CD31. A deep analysis of T cells with the CD4+CD25+CD127low/-CD26low/-CD45RAimmunophenotype revealed high expression of FoxP3 and/or CD39, while cells with the naïve immunophenotype, CD4+CD25+CD127low/-CD26low/-CD45RA+, presented lower expression of suppressor markers. Antigen CD31 is considered to be a valuable membrane marker of thymus-derived Tregs.
The presented 9-color panel that can be easily applied in laboratories enables reliable enumeration of Tregs with additional information about the functionality, maturity and origin of T regulatory cells.
调节性 T 细胞（Tregs）是一种独特的 CD4+T 细胞亚群，参与免疫反应的调节。传统上用于定义 Tregs 的免疫表型包括 CD4+CD25high 和转录因子叉头框蛋白 3（FoxP3）的表达。一种复杂的细胞内染色技术、激活的常规 T 淋巴细胞（Tcons）中 FoxP3 的瞬时上调以及通过下调 FoxP3 活性但具有去甲基化 FOXP3 启动子区域的幼稚 CD45RA+Tregs 的缺失可能导致不准确的定量。为了满足对可靠和简化计数策略的需求，我们研究了不同的膜标记物以捕获整个 Treg 区室并鉴定 Treg 的亚群。
在全血上进行分析。测试的门控策略基于以下膜抗原的表达：CD45、CD3、CD4、CD25、CD127、CD26、CD6、CD39、CD71、HLA-DR、CD45RA 和 CD31。进行了 FoxP3 的双对照。
最终的计数面板由膜标记物 CD45、CD3、CD4、CD25、CD127、CD26、CD39、CD45RA 和 CD31 组成。对具有 CD4+CD25+CD127low/-CD26low/-CD45RA 免疫表型的 T 细胞进行深入分析显示 FoxP3 和/或 CD39 的高表达，而具有幼稚免疫表型 CD4+CD25+CD127low/-CD26low/-CD45RA+的细胞表达较低的抑制性标志物。抗原 CD31 被认为是胸腺衍生 Tregs 的有价值的膜标记物。
本研究提出的 9 色面板易于在实验室中应用，可实现 Tregs 的可靠计数，并提供有关 T 调节细胞功能、成熟度和来源的额外信息。
