Soluble oligomeric TCR and antigen presentation to CTL

描述（由申请人提供）：越来越明显的是，沿着同源肽-MHC复合物（pMHC）的水平，它们在细胞表面上的分布以及与其他膜蛋白的结合也影响抗原呈递。我们已经表明，MHC I类分子（MHC-I）与粘附分子（ICAM-1）在膜筏和可溶性膜组分中共聚集，并且这些分子的生产性参与导致其额外招募到筏中并增加与Scr激酶的关联。靶细胞上筏完整性的破坏降低了病毒肽呈递给CTL的效力。我们相信，在CTL-靶细胞相遇期间，筏包括的MHC-I和ICAM-1的有效接合导致增强靶细胞向CTL呈递同源pMHC复合物的信号传导。在此基础上，我们将研究分子组装体对靶细胞和CTL的功能意义。我们希望确定膜和细胞内的蛋白质参与这些组件，并定义这些分子之间的“串扰”的机制。我们的努力得到了NIAID的5 RO 1 AI 52812 -01赠款的支持。到目前为止，该提案的一个重要方面还没有得到重视。也就是说，必须确定靶细胞上同源pMHC复合物的水平和分布。这需要一种试剂，该试剂可以将负载有感兴趣的肽的MHC分子与其他pMHC复合物区分开。特异于给定pMHC的可溶性TCR类似物可以用作探测靶细胞表面上的pMHC的优雅工具。由于TCR-pMHC反应的亲和力相对较低，可溶性TCR应寡聚化以增加结合的亲合力。我们已经表明，来自CTL克隆D3的对p17 Gag表位SLYNTVATL（SL 9）特异性的四聚体TCR可以特异性地检测靶细胞上SL 9-HLA-A2复合物的存在。然而，在较低的表位密度下，细胞表面上的pMHC复合物之间的距离对于四聚体来说可能太长而不能同时接合所有结合位点，导致其亲合力的损失。考虑到这一点，我们在此提出通过增加四聚体的化合价和/或四聚体结合位点之间的距离来产生其他形式的寡聚D3 TCR。我们将使用这些试剂来表征SL 9-HLA-A2复合物在用携带HIV Gag的重组牛痘病毒感染的靶细胞上的分布，并将确定该参数如何影响CTL应答的灵敏度和幅度。未来，这些新试剂将用于检测来自受感染个体的样本中的HIV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.