T细胞抗原识别非常有效:当扫描抗原呈递细胞(APC)的表面时,T细胞可以检测到只有几个单一的抗原肽/MHCs(pMHCs)的存在,结构相似的非刺激性内源性pMHCs通常远远超过它们(Irvine等人。,Nature419(6909):845-849,2002;Purbhoo等人.,NatImmunol5(5):524-530,2004;Huang等人。,免疫39(5):846-857,2013)。T细胞如何实现这一点仍然是神秘的,特别是考虑到TCR通常对抗原性pMHC具有相当中等的亲和力,至少在体外测量时(Davis等人,,AnnRevImmunol16:523-544,1998)。为了全面阐明这一点,我们开发了一种基于显微镜的检测方法,这使我们能够在原位定量TCR-pMHC相互作用,即,在T细胞的新生免疫突触的特殊范围内,该T细胞与用共刺激分子B7-1,粘附分子ICAM-1和pMHCs功能化的平面支持的脂质双层接触(Huppa等人。,自然463(7283):963-967,2010)(图。1).结合测量基于位点特异性标记的pMHC和TCR之间的Frster共振能量转移(FRET),用源自TCRβ反应性H57-597抗体的重组位点特异性标记的单链抗体片段(scFV)修饰(Huppa等人。,《自然》463(7283):963-967,2010)。FRET,量子力学现象,涉及两个相邻荧光团的偶极矩的非辐射耦合,供体分子和受体分子。FRET效率与染料间距离的6次幂成反比。因此,它可以用作分子标尺(Stryer和Haugland,自然课程,美国58(2):719-726,1967年)或,就像这里的情况一样,对适当标记的分子的相互作用进行评分。为了便于定量和单分子读出,以位点特异性方式缀合供体和受体染料是重要的。虽然SLB模拟了活细胞的质膜的一些但肯定不是全部特性,它们的使用具有许多操作优势:SLB可以在流体状态下制备,从而促进伴随免疫突触形成的空间重排(Grakoui等人。,科学285(5425):221-227,1999)。三维结合过程的成像减少到二维,这节省时间和荧光团发射的光子,并允许快速测量。此外,可以在噪声衰减全内反射(TIR)模式下获取图像,到目前为止,必须在免疫突触内进行单分子检测。重要的是,与细胞表面包埋的pMHCs相比,pMHCs的刺激效能得到了很好的保留。因此,虽然原则上是人造的,SLB仍然是T细胞在接近APC时遇到的生理情景的良好近似。反之亦然,重建方法提供了独特的机会,以高度定量的方式询问辅助分子对T细胞抗原识别的影响.在本章中,我们将提供用于SLB装饰的蛋白质生产的建议以及用于SLB生产的动手协议。我们将详细描述如何执行和分析基于FRET的实验以确定突触结合常数。在“注释”部分中,我们将提供一些有关显微镜设置的信息,以及数学和生物物理基础的基础数据分析。
T-cell antigen recognition is remarkably efficient: when scanning the surface of antigen-presenting cells (APCs), T-cells can detect the presence of just a few single antigenic peptide/MHCs (pMHCs), which are often vastly outnumbered by structurally similar non-stimulatory endogenous pMHCs (Irvine et al., Nature 419(6909):845-849, 2002; Purbhoo et al., Nat Immunol 5(5):524-530, 2004; Huang et al., Immunity 39(5):846-857, 2013). How T-cells achieve this is still enigmatic, in particular in view of the rather moderate affinity that TCRs typically exert for antigenic pMHCs, at least when measured in vitro (Davis et al., Ann Rev Immunol 16:523-544, 1998). To shed light on this in a comprehensive manner, we have developed a microscopy-based assay, which allows us to quantitate TCR-pMHC interactions in situ, i.e., within the special confines of the nascent immunological synapse of a T-cell contacting a planar-supported lipid bilayer functionalized with the costimulatory molecule B7-1, the adhesion molecule ICAM-1, and pMHCs (Huppa et al., Nature 463(7283):963-967, 2010) (Fig. 1). Binding measurements are based on Förster resonance energy transfer (FRET) between site-specifically labeled pMHCs and TCRs, which are decorated with recombinant site-specifically labeled single-chain antibody fragments (scFV) derived from the TCRβ-reactive H57-597 antibody (Huppa et al., Nature 463(7283):963-967, 2010). FRET, a quantum-mechanical phenomenon, involves the non-radiative coupling of dipole moments of two adjacent fluorophores, a donor molecule and an acceptor molecule. FRET efficiency is inversely proportional to the sixth power of the inter-dye distance. Hence, it can be employed as a molecular ruler (Stryer and Haugland, Proc Natl Acad Sci, USA 58(2):719-726, 1967) or, as is the case here, to score for interactions of appropriately labeled molecules. To facilitate both quantitative and single-molecule readout, it is important to conjugate donor and acceptor dyes in a site-specific manner.While SLBs mimic some but certainly not all properties of a plasma membrane of a living cell, their use features a number of operational advantages: SLBs can be prepared in a fluid state, thereby facilitating the spatial rearrangements that accompany the formation of an immunological synapse (Grakoui et al., Science 285(5425):221-227, 1999). The imaging of a three-dimensional binding process is reduced to two dimensions, which saves time and fluorophore-emitted photons and allows for fast measurements. Furthermore, images can be acquired in noise-attenuated total internal reflection (TIR) mode, so far a necessity for single-molecule detection within the immunological synapse. Importantly, the stimulatory potency of pMHCs is very well preserved compared to cell surface-embedded pMHCs. Hence, while in principle artificial, SLBs are still a good approximation of the physiologic scenario a T-cell encounters when approaching an APC. Vice versa, the reconstitutive approach offers unique opportunities to interrogate the influence of accessory molecules on T-cell antigen recognition in a highly quantitative manner.In this chapter we will provide recommendations for the production of proteins used for SLB decoration as well as hands-on protocols for the production of SLBs. We will describe in detail how to perform and analyze FRET-based experiments to determine synaptic binding constants. In the \"Notes\" section, we will provide some information regarding the microscope setup as well as the mathematical and biophysical foundation underlying data analysis.