Measuring TCR-pMHC Binding In Situ using a FRET-based Microscopy Assay
Measuring TCR-pMHC Binding In Situ using a FRET-based Microscopy Assay
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DOI：
10.3791/53157
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发表时间：
2015-10-01
影响因子：
1.2
通讯作者：
Huppa, Johannes B.
中科院分区：
文献类型：
--
作者：
Axmann, Markus;Schuetz, Gerhard J.;Huppa, Johannes B.
T细胞在识别抗原呈递细胞（APC）表面上的MHC分子（pMHC）中嵌入的肽形式的抗原时是非常特异和有效的。尽管在体外测量结合时，T细胞抗原受体（TCR）通常对抗原产生中等亲和力（μ M范围）（1）。鉴于有助于T细胞抗原敏感性的分子和细胞参数，已经开发了一种基于显微镜的方法，作为原位监测TCR-pMHC结合的手段，因为它发生在活T细胞和人工和功能化玻璃支撑的平面脂质双层（SLB）的突触内，其模拟抗原呈递细胞（APC）的细胞膜（2）。测量基于附着于TCR和pMHC的蓝移和红移荧光染料之间的福斯特共振能量转移（FRET）。因为FRET的效率与染料间距离的六次方成反比，所以可以采用FRET信号来可视化突触TCR-pMHC结合。显微镜方法的灵敏度支持单分子FRET事件的检测。这允许确定突触TCR-pMHC相互作用的亲和力和解离速率，进而内插结合的结合速率。类似的测定可应用于测量其天然环境中的其他受体-配体相互作用。
T-cells are remarkably specific and effective when recognizing antigens in the form of peptides embedded in MHC molecules (pMHC) on the surface of Antigen Presenting Cells (APCs). This is despite T-cell antigen receptors (TCRs) exerting usually a moderate affinity (mu M range) to antigen when binding is measured in vitro(1). In view of the molecular and cellular parameters contributing to T-cell antigen sensitivity, a microscopy-based methodology has been developed as a means to monitor TCR-pMHC binding in situ, as it occurs within the synapse of a live T-cell and an artificial and functionalized glass-supported planar lipid bilayer (SLB), which mimics the cell membrane of an Antigen presenting Cell (APC) (2). Measurements are based on Forster Resonance Energy Transfer (FRET) between a blue-and red-shifted fluorescent dye attached to the TCR and the pMHC. Because the efficiency of FRET is inversely proportional to the sixth power of the inter-dye distance, one can employ FRET signals to visualize synaptic TCR-pMHC binding. The sensitive of the microscopy approach supports detection of single molecule FRET events. This allows to determine the affinity and off-rate of synaptic TCR-pMHC interactions and in turn to interpolate the on-rate of binding. Analogous assays could be applied to measure other receptor-ligand interactions in their native environment.