Medical Faculty, Institute of Clinical Immunology, University of Leipzig, Leipzig, Germany.
Department of Pediatrics, Jena University Hospital, Jena, Germany.
Front Immunol. 2019 Apr 5;10:722. doi: 10.3389/fimmu.2019.00722. eCollection 2019.
Here we present a simple and sensitive flow cytometric-based assay to assess T cell proliferation. Given the critical role STAT5A phosphorylation in T cell proliferation, we decided to evaluate phosphorylation of STAT5A as an indicator of T cell proliferation. We determined pSTAT5A in T cell treated with either CD3/CD28 or PHA. After stimulation, T cells from adult healthy donors displayed a strong long-lasting phosphorylation of STAT5A, reaching a peak value after 24 h. The median fluorescence intensity (MFI) of pSTAT5A increased from 112 ± 17 to 512 ± 278 (CD3/CD28) (24 h) and to 413 ± 123 (PHA) (24 h), the IL-2 receptor-α (CD25) expression was greatly enhanced and after 72 h T cell proliferation amounted to 52.3 ± 10.3% (CD3/CD28) and to 48.4 ± 9.7% (PHA). Treatment with specific JAK3 and STAT5 inhibitors resulted in a complete blockage of phosphorylation of STAT5A, CD25 expression, and suppression of T cell proliferation. Compared with currently available methods, STAT5A phosphorylation is well-suited to predict T cell proliferation. Moreover, the method presented here is not very time consuming (several hours) and delivers functional information from which conclusions about T cell proliferation can be drawn.
在这里，我们提出了一种简单而敏感的流式细胞术检测方法，用于评估 T 细胞增殖。鉴于 STAT5A 磷酸化在 T 细胞增殖中的关键作用，我们决定将 STAT5A 磷酸化作为 T 细胞增殖的指标进行评估。我们在经 CD3/CD28 或 PHA 处理的 T 细胞中测定了 pSTAT5A。刺激后，来自成年健康供体的 T 细胞显示出强烈的持续 STAT5A 磷酸化，在 24 小时后达到峰值。pSTAT5A 的中荧光强度（MFI）从 112±17 增加到 512±278（CD3/CD28）（24 小时）和 413±123（PHA）（24 小时），白细胞介素-2 受体-α（CD25）的表达大大增强，72 小时后 T 细胞增殖达到 52.3±10.3%（CD3/CD28）和 48.4±9.7%（PHA）。用特异性 JAK3 和 STAT5 抑制剂处理可完全阻断 STAT5A 磷酸化、CD25 表达，并抑制 T 细胞增殖。与目前可用的方法相比，STAT5A 磷酸化非常适合预测 T 细胞增殖。此外，这里提出的方法并不非常耗时（几个小时），并提供了可从中得出关于 T 细胞增殖的结论的功能信息。
