Institute of Clinical Immunology, Medical Faculty, University of Leipzig, Leipzig, Germany.
Department of Pediatrics, Dr. von Hauner Children's Hospital, University Hospital, Ludwig-Maximilians-University of Munich, Munich, Germany.
Cytometry A. 2021 Aug;99(8):774-783. doi: 10.1002/cyto.a.24286. Epub 2020 Dec 20.
Cytomegalovirus (CMV)-specific T cells expand with CMV reactivation and are probably prerequisite for control and protection. Given the critical role STAT5A phosphorylation (pSTAT5A) in T cell proliferation, this study presents a simple and sensitive flow cytometric-based pSTAT5A assay to quickly identify CMV-specific T cell proliferation. We determined pSTAT5A in T cells treated with CMV-specific peptide mix (pp65 + IE1 peptides) from 20 healthy adult subjects and three immunodeficient patients with CARMIL-2 mutation. After stimulation, the percentage of pSTAT5A T cells in CMV-seropositive (CMV ) subjects significantly increased from 3.0% ± 1.9% (unstimulated) to 11.4% ± 5.9% (stimulated) for 24 h. After 7 days of stimulation, the percentage of expanded T cells amounted to 26% ± 17.2%. Conversely, the percentage of pSTAT5A T cells and T cell proliferation from CMV-seronegative (CMV ) subjects hardly changed (from 3.0% ± 1.3% to 3.7% ± 1.8% and from 4.3% ± 2.1% to 5.7% ± 1.7%, respectively). We analyzed the correlation between the percentage of pSTAT5A T cells versus (1) CMV-IgG concentrations versus (2) the percentage of expanded T cells and versus (3) the percentage of initial CMV-specific T cells. In immunodeficient patients with CARMIL-2 mutation, CMV-specific pSTAT5A and T cell proliferation were completely deficient. In conclusion, flow cytometric-based pSTAT5A assay represents an appropriate tool to quickly identify CMV-specific T cell proliferation and helps to understand dysfunctions in controlling other pathogens. Flow cytometric-based pSTAT5A assay may be a useful test in clinical practice and merits further validation in large studies.
巨细胞病毒 (CMV)-特异性 T 细胞随着 CMV 再激活而扩增，可能是控制和保护的前提。鉴于 STAT5A 磷酸化 (pSTAT5A) 在 T 细胞增殖中的关键作用，本研究提出了一种简单而灵敏的基于流式细胞术的 pSTAT5A 检测方法，可快速识别 CMV 特异性 T 细胞增殖。我们测定了来自 20 名健康成年受试者和 3 名 CARMIL-2 突变免疫缺陷患者的 CMV 特异性肽混合物（pp65+IE1 肽）处理后的 T 细胞中的 pSTAT5A。刺激后，CMV 阳性（CMV+）受试者的 pSTAT5A T 细胞百分比从刺激前的 3.0%±1.9%显著增加至 24 小时后的 11.4%±5.9%。刺激 7 天后，扩增的 T 细胞百分比达到 26%±17.2%。相反，CMV 阴性（CMV-）受试者的 pSTAT5A T 细胞百分比和 T 细胞增殖几乎没有变化（从 3.0%±1.3%变为 3.7%±1.8%，从 4.3%±2.1%变为 5.7%±1.7%）。我们分析了 pSTAT5A T 细胞百分比与（1）CMV-IgG 浓度，（2）扩增的 T 细胞百分比和（3）初始 CMV 特异性 T 细胞百分比之间的相关性。在 CARMIL-2 突变的免疫缺陷患者中，CMV 特异性 pSTAT5A 和 T 细胞增殖完全缺乏。总之，基于流式细胞术的 pSTAT5A 检测是一种快速识别 CMV 特异性 T 细胞增殖的合适工具，有助于了解控制其他病原体的功能障碍。基于流式细胞术的 pSTAT5A 检测可能是临床实践中的有用测试，值得在大型研究中进一步验证。
