CHEMOKINE REGULATION OF NKT CELLS, AND ACAID

趋化因子与多种炎症性和自身免疫性疾病有关，因为它们在免疫诱导和炎症部位募集细胞。然而，很少有研究探索趋化因子在外周耐受诱导中的作用，也没有关于NKT细胞的特异性趋化因子的研究报道。这项研究计划将探索特定的趋化因子在免疫炎症反应的负面调节中的作用，特别是在NKT细胞招募到耐受诱导部位的作用。前房相关性免疫偏离(ACAID)与眼内抗原输入后对迟发型超敏反应(DTH)的选择性负调节有关。脾NKT细胞的聚集对于ACAID的发展是绝对必要的。初步数据显示，MIP-2与NKT细胞募集有关。我们将使用分子、细胞和形态技术来鉴定和确定趋化因子在耐受诱导过程中的作用。目的通过核糖核酸酶保护试验和多色流式细胞术确定趋化因子的表达谱。第二个目标将使用经典的趋化试验来确定NKT细胞对ACAID相关趋化因子的迁移反应，以及确认这些细胞上趋化因子受体的表达的分子技术。第三个目的是通过将趋化因子受体基因敲除小鼠的NKT细胞重组为NKT细胞敲除小鼠，探讨在ACAID诱导过程中趋化因子与NKT细胞相互作用的机制。局部过继转移试验将测量是否产生了调节性T细胞。最后，第四个目标将探索这样一个假设，即为诱导ACAID而招募到脾的细胞必须形成相互作用的细胞团。在确定这样的簇和其中的细胞后，我们将调节趋化因子和潜在的黏附分子，并通过簇的质量和数量来评估结果。NKT细胞缺陷和/或缺陷与小鼠和人类的各种自身免疫疾病(狼疮、1型糖尿病、系统性硬皮病)有关。因此，NKT细胞介导的T调节细胞的诱导机制可能在维持多种器官和组织的自身耐受性方面发挥作用，除了眼睛等免疫特权部位外。这项提议中提出的关于趋化因子、先天细胞和外周耐受的新假设，代表了迄今为止完全未被探索的研究领域。

Chemokines are associated with a variety of inflammatory and autoimmune diseases for their role in recruitment of cells to the sites of immune induction and inflammation. However, few studies have explored a role for chemokines in peripheral tolerance induction and no studies are reported for specific chemokines for NKT cells. This research plan will explore the role of specific chemokines in negative regulation of an immune inflammatory response and specifically in the recruitment of NKT cells to the site of tolerance induction. Anterior Chamber Associated Immune Deviation (ACAID) is associated with the selective negative regulation of delayed type hypersensitivity (DTH) after the introduction of antigen into the eye. An accumulation of splenic NKT cells is absolutely required for the development of ACAID. Preliminary data show that MIP-2 is associated with NKT cell recruitment. We will use molecular, cellular and morphological techniques to identify and define the role of the chemokines in the tolerance inducing process. Aim One will define the chemokine profile by RNase Protection Assay and multi-color flow cytometry. The second aim will use classic chemotaxis assays to define the migratory response of NKT cells to ACAID associated chemokines, along with molecular techniques to confirm the expression of chemokine receptors on these cells. The third aim will explore the mechanisms of chemokine-NKT cell interaction during ACAID induction by reconstituting NKT cell knockout mice with NKT cells from chemokine receptor knockout mice. A local adoptive transfer assay will measure if regulator T cells were generated. Finally, the fourth aim will explore the hypothesis that the cells recruited to the spleen for the induction of ACAID must necessarily form interactive cell clusters. After identifying such clusters and cells within, we will modulate chemokines and potential adhesion molecules and evaluate the outcome by quality and quantity of clusters. NKT cell defects and/or deficiency is associated with a variety of autoimmune disorders in both mice and humans (lupus, type 1 diabetes, systemic scleroderma). Thus, the mechanisms of NKT cell mediated induction of T regulatory cells might function in the maintenance of self tolerance in a variety of organs and tissues in addition to immune privileged sites such as the eye. The novel postulates about chemokines, innate cells and peripheral tolerance presented in this proposal, represent a heretofore totally unexplored area of research.