Soluble oligomeric TCR and antigen presentation to CTL

描述(申请人提供)：越来越明显的是，随着同源肽-MHC复合体(PMHC)的水平，它们在细胞表面的分布以及与其他膜蛋白的结合也影响抗原呈递。我们已经证明，MHC-I类分子(MHC-I)与黏附分子(ICAM-1)在膜筏和可溶性膜组分中都是共同聚集的，这些分子的生产性接触导致它们更多地被募集到膜筏中，并增加与SCR激酶的结合。靶细胞RAFT完整性的破坏降低了病毒多肽呈递给CTL的效力。我们认为，在CTL-靶细胞接触过程中，包含RAFT的MHC-I和ICAM-1的生产性接触导致了一种信号，该信号增强了靶细胞对CTL的同源pMHC复合体的呈递。在此基础上，我们开始研究靶细胞和CTL上分子组装的功能意义。我们希望确定参与这些组装的细胞膜和细胞内蛋白质，并确定这些分子之间的“串扰”机制。我们的努力得到了NIAID的5 RO1 AI52812-01拨款的支持。到目前为止，该提案的一个重要方面一直被遗漏。也就是说，确定同源pMHC复合体在靶细胞上的水平和分布是至关重要的。这需要一种试剂，能够将负载了感兴趣的多肽的MHC分子与其他pMHC复合体区分开来。针对特定pMHC的可溶性TCR类似物可以作为探测靶细胞表面pMHC的一种很好的工具。由于TCR-pMHC反应的亲和力相对较低，应对可溶性TCR进行齐聚以增加结合的亲和力。我们已经证明，针对p17 GAG表位SLYNTVATL(SL9)的CTL克隆D3的四聚体TCR可以特异性地检测靶细胞上SL9-HLA-A2复合体的存在。然而，在较低的表位密度下，细胞表面pMHC复合体之间的距离可能太长，以至于四聚体无法同时与所有结合位点结合，从而失去了亲和力。考虑到这一点，我们建议在这里通过增加四聚体的价态和/或四聚体结合位点之间的距离来产生其他形式的寡聚体D3TCR。我们将使用这些试剂来表征SL9-HLA-A2复合体在感染携带HIV Gag的重组痘苗病毒感染的靶细胞上的分布，并确定这一参数如何影响CTL反应的敏感性和大小。未来，这些新试剂将用于检测从感染者身上提取的样本中的艾滋病毒感染细胞。

DESCRIPTION (provided by applicant): It is becoming increasingly evident that along with the level of cognate peptide-MHC complexes (pMHC) their distribution on the cell surface as well as association with other membrane proteins also influences the antigen presentation. We have shown that MHC class I molecules (MHC-I) are co-clustered with adhesion molecules (ICAM-1) in both membrane rafts and soluble membrane fraction and that productive engagement of these molecules leads to their additional recruitment into rafts and increased association with Scr kinases. Disruption of the raft integrity on target cells decreases potency of viral peptides presentation to CTL. We believe that productive engagement of raft-included MHC-I and ICAM-1 during CTL-target cell encounter results in a signaling that augment presentation of cognate pMHC complexes by target cells to CTL. Based on this, we are set up to investigate functional significance of molecular assemblies on target cells and CTL. We want to identify membrane and intracellular proteins involving in these assemblies and to define mechanisms of "cross-talk" between these molecules. Our efforts are supported by 5 RO1 AI52812-01 Grant from NIAID. One important aspect of the proposal has been missing thus far. Namely, it is essential to determine the level and distribution of cognate pMHC complexes on target cells. This requires a reagent that can distinguish MHC molecules loaded with a peptide of interest from other pMHC complexes. A soluble TCR analog specific for a given pMHC may serve as an elegant tool to probe the pMHC on the surface of target cells. Because of the relatively low affinity of the TCR-pMHC reaction, soluble TCR should be oligomerized to increase the avidity of the binding. We have shown that tetrameric TCR from CTL clone D3 specific for p17 Gag epitope SLYNTVATL (SL9) can specifically detect the presence of SL9-HLA-A2 complexes on target cells. However, at lower epitope density the distance between the pMHC complexes on the cell surface may be too long for the tetramer to engage all binding sites at the same time resulting in a loss of its avidity. With this in mind, we propose here to produce other forms of oligomeric D3 TCR by increasing the valency of the tetramer and/or the distance between tetramer binding sites. We will use these reagents to characterize the distribution of SL9-HLA-A2 complexes on target cells infected with recombinant Vaccinia virus carrying HIV Gag and will determine how this parameter influences sensitivity and magnitude of CTL responses. In the future, these new reagents will be used to detect HIV infected cells in samples derived from infected individuals.