Immune receptors on Cytotoxic Lymphocytes& Target Cell

描述（由申请人提供）：尽管在某些病毒感染的细胞中抗原肽的天然丰度非常低，但这些细胞仍被病毒特异性细胞毒性T淋巴细胞（CTL）裂解。我们已经发现，靶细胞上少于12个同源肽-MHC（pMHC）复合物足以使它们对CTL的特异性裂解敏感。CTL对靶细胞裂解的显著敏感性的分子机制尚不清楚。我们的初步数据表明，释放少量的细胞溶解颗粒不会诱导靶细胞溶解，除非颗粒集中在CTL和靶细胞膜之间的有限空间。我们假设免疫突触将颗粒集中在精确的位置保持其活性，并解释了CTL裂解靶细胞的敏感性和特异性。我们还发现，LFA- 1 - 1CAM- 1相互作用对于CTL形成免疫突触的早期阶段是足够的。这让我们提出，通过粘附分子的信号传导导致形成具有两个典型结构域或超分子激活簇（SMAC）的瞬时突触。瞬时突触可能有助于CTL有效扫描靶细胞表面。最近，我们发现MHC-I和ICAM-1分子在生物化学上和通过膜筏内成像而共定位。我们还表明，筏的完整性有利于病毒肽-MHC（pMHC）的CTL检测通过机制，需要LFA-1和ICAM-1的相互作用。基于此，我们假设筏中的ICAM-1-MHC-I共聚集提供了CTL上的早期免疫突触形成与靶细胞上的抗原呈递之间的联系。我们进一步假设MHC-I和ICAM-1通过衔接蛋白被募集到筏上，形成复杂的分子组装体，这些分子组装体在不同的细胞类型上可能具有不同的结构。使用具有已知特异性的人CTL克隆的精确操作，我们将通过追求3个特定目标来测试该假设：（i）通过研究在T细胞和靶细胞膜之间的界面处的细胞溶解颗粒释放以及LFA-1-ICAM-1的作用，确定免疫突触的细胞溶解分子浓度的意义。（ii）通过研究粘附分子模式和这些模式的功能相关性，确定CTL和NK细胞形成免疫突触的机制;（iii）确定膜筏中MHC-I和ICAM-1相互作用的功能重要性和机制。

DESCRIPTION (provided by applicant):Although natural abundance of antigenic peptides in some virus-infected is very low, these cells are still lysed by virus-specific cytotoxic T lymphocytes (CTL). We have found that less then a dozen cognate peptide-MHC (pMHC) complexes on target cells are sufficient to render them susceptible for specific lysis by CTL. The molecular mechanism accounting for the striking sensitivity of target cell lysis by CTL is not understood. Our preliminary data suggest that release of a small amount of cytolytic granules would not induce target cell lysis unless the granules are concentrated in a limited space between CTL and target cell membranes. We hypothesized that immunological synapse concentrates the granules preserving their activity at the precise location and accounts for the sensitivity and specificity of target cell lysis by CTL. We have also found that LFA- 1 -1CAM- 1 interactions are sufficient for early stages of immunological synapse formation by CTL. This let us to propose that signaling through adhesion molecules results in the formation of transient synapse that has two typical domains or supramolecular activation clusters (SMACs). The transient synapse is likely to facilitate effective scanning of the surface of target cells by CTL. Most recently, we have found that MHC-I and ICAM-1 molecules are co-localized biochemically and by imaging within membrane rafts. We also showed that raft integrity facilitates viral peptide-MHC (pMHC) detection by CTL through mechanisms that require interaction of LFA-1 and ICAM-1. Based on this we hypothesized that ICAM-1-MHC-I co- clustering in rafts provides the linkage between early immunological synapse formation on CTL and the presentation of antigen on target cells. We further hypothesize that MHC-I and ICAM-1 are recruited to the rafts through adapter proteins to form complex molecular assemblies, which might have distinct structures on various cell types. Using precise manipulation of human CTL clones with known specificity, we will test this hypothesis by pursuing 3 specific aims: (i) To determine the significance of cytolytic molecule concentration by the immunological synapses by studying cytolytic granule release at the interface between the T cell and target cell membranes and the role of LFA-1-ICAM-1 interactions that lead to the synapse formation and effective target cell lysis by CTL; (ii) To determine the mechanism of immunological synapse formation by CTL and NK cells studying adhesion molecule patterns and functional correlates of these patterns; (iii) To determine the functional importance and mechanism of MHC-I and ICAM-1 interactions in membrane rafts.