Translational and Clinical Research Program, Centro de Investigación del Cáncer (CIC) and Instituto de Biología Molecular y Celular del Cancer (IBMCC), CSIC-University of Salamanca (USAL), Salamanca, Spain.
Cytometry Service, NUCLEUS, Department of Medicine, University of Salamanca (USAL) and Institute of Biomedical Research of Salamanca (IBSAL), Salamanca, Spain.
Front Immunol. 2020 Feb 27;11:166. doi: 10.3389/fimmu.2020.00166. eCollection 2020.
CD4+ T cells comprise multiple functionally distinct cell populations that play a key role in immunity. Despite blood monitoring of CD4+ T-cell subsets is of potential clinical utility, no standardized and validated approaches have been proposed so far. The aim of this study was to design and validate a single 14-color antibody combination for sensitive and reproducible flow cytometry monitoring of CD4+ T-cell populations in human blood to establish normal age-related reference values and evaluate the presence of potentially altered profiles in three distinct disease models-monoclonal B-cell lymphocytosis (MBL), systemic mastocytosis (SM), and common variable immunodeficiency (CVID). Overall, 145 blood samples from healthy donors were used to design and validate a 14-color antibody combination based on extensive reagent testing in multiple cycles of design-testing-evaluation-redesign, combined with functional studies, gene expression profiling, and multicentric evaluation of manual vs. automated gating. Fifteen cord blood and 98 blood samples from healthy donors (aged 0-89 years) were used to establish reference values, and another 25 blood samples were evaluated for detecting potentially altered CD4 T-cell subset profiles in MBL ( = 8), SM ( = 7), and CVID ( = 10). The 14-color tube can identify ≥89 different CD4+ T-cell populations in blood, as validated with high multicenter reproducibility, particularly when software-guided automated (vs. manual expert-based) gating was used. Furthermore, age-related reference values were established, which reflect different kinetics for distinct subsets: progressive increase of naïve T cells, T-helper (Th)1, Th17, follicular helper T (TFH) cells, and regulatory T cells (Tregs) from birth until 2 years, followed by a decrease of naïve T cells, Th2, and Tregs in older children and a subsequent increase in multiple Th-cell subsets toward late adulthood. Altered and unique CD4+ T-cell subset profiles were detected in two of the three disease models evaluated (SM and CVID). In summary, the EuroFlow immune monitoring TCD4 tube allows fast, automated, and reproducible identification of ≥89 subsets of CD4+ blood T cells, with different kinetics throughout life. These results set the basis for in-depth T-cell monitoring in different disease and therapeutic conditions.
CD4+ T 细胞包含多个具有不同功能的细胞群，在免疫中起着关键作用。尽管对 CD4+ T 细胞亚群的血液监测具有潜在的临床应用价值，但迄今为止尚未提出标准化和经过验证的方法。本研究旨在设计和验证一种单一的 14 色抗体组合，用于敏感且可重复地监测人血液中的 CD4+ T 细胞群，以建立正常的年龄相关参考值，并评估三种不同疾病模型中单克隆 B 细胞淋巴增生症（MBL）、系统性肥大细胞增多症（SM）和常见可变免疫缺陷（CVID）中潜在改变的特征。总体而言，使用 145 份来自健康供体的血液样本，通过在多个设计-测试-评估-重新设计循环中进行广泛的试剂测试，结合功能研究、基因表达谱分析以及手动与自动门控的多中心评估，设计和验证了一种基于 14 种颜色的抗体组合。使用 15 份脐带血和 98 份来自健康供体（年龄 0-89 岁）的血液样本建立参考值，另外 25 份血液样本用于检测 MBL（=8）、SM（=7）和 CVID（=10）中潜在改变的 CD4 T 细胞亚群特征。14 色管可在血液中鉴定出≥89 种不同的 CD4+ T 细胞群，其具有高度的多中心可重复性，特别是当使用软件指导的自动（与基于专家的手动）门控时。此外，还建立了年龄相关的参考值，反映了不同亚群的不同动力学：从出生到 2 岁，幼稚 T 细胞、辅助性 T 细胞（Th）1、Th17、滤泡辅助性 T（TFH）细胞和调节性 T 细胞（Treg）的数量逐渐增加，然后幼稚 T 细胞、Th2 和 Treg 的数量在年龄较大的儿童中减少，随后多个 Th 细胞亚群在成年后期增加。在评估的三种疾病模型中的两种（SM 和 CVID）中检测到改变和独特的 CD4+ T 细胞亚群特征。总之，EuroFlow 免疫监测 TCD4 管可快速、自动和可重复地识别血液中≥89 种 CD4+ T 细胞亚群，其动力学在整个生命周期中都有所不同。这些结果为在不同疾病和治疗条件下进行深入的 T 细胞监测奠定了基础。
