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 in vitro 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 (n = 8), SM (n = 7), and CVID (n = 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 细胞包含多种功能不同的细胞群，在免疫中发挥关键作用。尽管 CD4+ T 细胞亚群的血液监测具有潜在的临床实用性，但迄今为止尚未提出标准化和经过验证的方法。本研究的目的是设计和验证单一 14 色抗体组合，用于对人血液中 CD4+ T 细胞群进行灵敏且可重复的流式细胞术监测，以建立正常的年龄相关参考值，并评估血液中是否存在潜在改变的特征。三种不同的疾病模型——单克隆 B 细胞淋巴细胞增多症 (MBL)、系统性肥大细胞增多症 (SM) 和常见变异型免疫缺陷症 (CVID)。总体而言，基于设计-测试-评估-重新设计多个周期中的广泛试剂测试，结合体外功能研究、基因表达谱和多中心，使用来自健康捐献者的 145 份血液样本来设计和验证 14 色抗体组合评估手动与自动门控。使用来自健康捐献者（年龄 0-89 岁）的 15 份脐带血和 98 份血液样本来建立参考值，并评估另外 25 份血液样本，以检测 MBL (n = 8)、SM 中潜在改变的 CD4 T 细胞亚群谱(n = 7) 和 CVID (n = 10)。 14 色管可以识别血液中 ≥89 个不同的 CD4+ T 细胞群，经多中心重复性验证，特别是在使用软件引导的自动门控（相对于基于专家的手动门控）时。 此外，还建立了与年龄相关的参考值，反映了不同亚群的不同动力学：幼稚 T 细胞、辅助性 T (Th)1、Th17、滤泡辅助性 T (TFH) 细胞和调节性 T 细胞 (Treg) 的逐渐增加从出生到 2 岁，年龄较大的儿童中幼稚 T 细胞、Th2 和 Tregs 会减少，而到成年后期，多种 Th 细胞亚群会增加。在评估的三种疾病模型中的两种（SM 和 CVID）中检测到改变的和独特的 CD4+ T 细胞亚群谱。总之，EuroFlow 免疫监测 TCD4 管可以快速、自动化和可重复地识别 ≥89 个 CD4+ 血 T 细胞亚群，并且在整个生命周期中具有不同的动力学。这些结果为不同疾病和治疗条件下的深入 T 细胞监测奠定了基础。