Immune receptors on Cytotoxic Lymphocytes& Target Cell

描述(申请人提供)：尽管一些病毒感染者体内抗原肽的自然丰度很低，但这些细胞仍由病毒特异性细胞毒性T淋巴细胞(CTL)裂解。我们发现靶细胞上不到12个同源肽-MHC(PMHC)复合体足以使它们容易被CTL特异性裂解。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，释放少量的溶细胞颗粒不会诱导靶细胞溶解，除非这些颗粒集中在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准确的位置，保持其活性，并解释了CTL对靶细胞裂解的敏感性和特异性。我们还发现，LFA-1-1CAM-1的相互作用对于CTL免疫突触形成的早期阶段是足够的。这使得我们提出，通过黏附分子传递信号导致了具有两个典型结构域或超分子激活簇(SMAC)的瞬时突触的形成。这种瞬时突触可能有助于CTL对靶细胞表面的有效扫描。最近，我们发现MHC-I和ICAM-1分子通过生物化学和成像技术共同定位于膜筏内。我们还发现RAFT的完整性通过需要LFA-1和ICAM-1相互作用的机制促进了CTL对病毒多肽-MHC(PMHC)的检测。在此基础上，我们假设ICAM-1-MHC-I在RAFT中的共同聚集在CTL上的早期免疫突触形成和靶细胞上的抗原提呈之间提供了联系。我们进一步假设，MHC-I和ICAM-1通过适配器蛋白被招募到筏子上，形成复杂的分子组件，这些组件可能在不同类型的细胞上具有不同的结构。我们将使用具有已知特异性的人CTL克隆的精确操作，通过追求三个特定目标来验证这一假说：(I)通过研究T细胞和靶细胞膜之间的溶细胞颗粒释放以及LFA-1-ICAM-1相互作用导致突触形成和CTL有效的靶细胞溶解作用，确定免疫突触产生的细胞溶解分子浓度的意义；(Ii)确定CTL和NK细胞免疫突触形成的机制，研究这些模式的黏附分子模式和功能相关性；(Iii)确定MHC-I和ICAM-1相互作用在膜上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.