Real-time analysis of T cell receptors in naive cells in vitro and in vivo reveals flexibility in synapse and signaling dynamics
Real-time analysis of T cell receptors in naive cells in vitro and in vivo reveals flexibility in synapse and signaling dynamics
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DOI：
10.1084/jem.20091201
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发表时间：
2010-11-22
影响因子：
15.3
通讯作者：
Krummel, Matthew F.
中科院分区：
文献类型：
--
作者：
Friedman, Rachel S.;Beemiller, Peter;Krummel, Matthew F.
T细胞受体（TCR）的实时动态反映了抗原检测和T细胞信号传导，为免疫应答的演变事件提供了有价值的见解。尽管在体外简化系统中研究TCR动力学方面取得了相当大的进展，但在完整组织中以生理密度表达的亚细胞信号传导复合物的实时成像一直具有挑战性。在这项研究中，我们产生了一个转基因小鼠的TCR融合到绿色荧光蛋白，以提供深入了解免疫反应的早期信号事件。为了能够成像淋巴结中的初始T细胞中的TCR动力学，我们增强了荧光TCR融合蛋白的信号检测，并使用第二荧光团的体积掩蔽来标记表达荧光TCR的T细胞。这些体内分析和体外平行实验显示TCR最小和瞬时掺入到稳定的中央超分子活化簇（cSMAC）结构中，但强有力的证据表明快速的抗原依赖性TCR内化不依赖于T细胞运动性停滞或cSMAC形成。也偶尔观察到短寿命的抗原非依赖性TCR聚集。这些体内观察结果表明，在早期T细胞活化期间发生不同的TCR运输和细胞停滞动力学。
The real-time dynamics of the T cell receptor (TCR) reflect antigen detection and T cell signaling, providing valuable insight into the evolving events of the immune response. Despite considerable advances in studying TCR dynamics in simplified systems in vitro, live imaging of subcellular signaling complexes expressed at physiological densities in intact tissues has been challenging. In this study, we generated a transgenic mouse with a TCR fused to green fluorescent protein to provide insight into the early signaling events of the immune response. To enable imaging of TCR dynamics in naive T cells in the lymph node, we enhanced signal detection of the fluorescent TCR fusion protein and used volumetric masking with a second fluorophore to mark the T cells expressing the fluorescent TCR. These in vivo analyses and parallel experiments in vitro show minimal and transient incorporation of TCRs into a stable central supramolecular activating cluster (cSMAC) structure but strong evidence for rapid, antigen-dependent TCR internalization that was not contingent on T cell motility arrest or cSMAC formation. Short-lived antigen-independent TCR clustering was also occasionally observed. These in vivo observations demonstrate that varied TCR trafficking and cell arrest dynamics occur during early T cell activation.