The role of dendritic cell-T cell interactions in the p*

树突状细胞-T细胞相互作用在p*中的作用

• 批准号: 6932041
• 负责人: Mariana J Kaplan
• 金额: $ 7.65万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-08-03 至 2007-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6932041
• 关键词: T lymphocyte; apoptosis; cell cell interaction; cell differentiation; cell proliferation; cellular polarity; clinical research; cytotoxicity; dendritic cells; disease /disorder etiology; enzyme linked immunosorbent assay; flow cytometry; fluorescence microscopy; human subject; immunopathology; mixed tissue /cell culture; monoclonal antibody; monocyte; necrosis; phagocytosis; polymerase chain reaction; rheumatoid arthritis; systemic lupus erythematosus

DESCRIPTION (provided by applicant): Hypothesis: In systemic lupus erythematosus (SLE) differentiation and maturation of myeloid dendritic cells (My-DC) are impaired and play an important role in the development of abnormal T cell (TC) responses that contribute to the pathogenesis of the disease. Background and rationale: DCs are key regulators of the immune system and potent TC stimulators. It has been proposed that systemic autoimmune responses that characterize SLE could be explained by alterations in DC function. However, information is needed regarding whether DCs from SLE patients have abnormal differentiation, maturation and abnormal interactions with autologous TCs and the role that these abnormalities could play in the pathogensis of SLE. Specific aims: 1. Compare phenotypic and functional parameters in monocyte derived DCs from SLE patients and controls 2. Determine the specific characteristics of TC-DC interactions in SLE patients when compared to controls, including cell proliferation, differentiation/maturation and death. Methods: A) DC maturation and differentiation in response to specific stimuli will be evaluated in SLE patients and healthy controls by flow cytometry and multiplex RT-PCR. DC sensitivity to different apoptotic stimuli will be compared in both groups with Annexin V/Propidium iodide staining. B) Capacity of SLE and control DCs to phagocytose and internalize apoptotic and necrotic cells will be evaluated by DC incorporation of CMFDA-labeled apoptotic or necrotic cells using flow cytometry and fluorescent microscopy. Phagocytic capacity will also be evaluated using FITC-dextran. C) Cytokine production by SLE and control monocyte-derived DCs will be determined using multiplex ELISA and results confirmed by RT-PCR. D) DC-TC interactions will be addressed by determining apoptosis susceptibility in co-culture of both cell types and T cell subsets using flow cytometry, cytotoxicity assays and inhibition assays with specific monoclonal antibodies. E) The role of autologous DCs in inducing T cell proliferation and activation of specific TC subsets in SEE patients will be assessed by flow cytometry and H3 incorporation. F) The role of SLE DCs in inducing TH1/TH2 polarization will be evaluated by cytometric bead array. Significance: The results of the studies proposed in this grant might identify potential mechanisms involved in the generation of autoimmune responses and TC abnormalities in SLE. In addition, a better understanding of the molecular basis of DC differentiation and maturation in SLE may provide conceptual tools to regulate DC maturation and/or DC migration to lymphoid organs which may limit excessive stimulation of autoreactive T cells. These could lead into the development of novel therapeutic interventions designed to reverse these abnormalities and abrogate or block the onset and/or severity of this disease.

描述(申请人提供)：假设：在系统性红斑狼疮(SLE)中，髓系树突状细胞(My-DC)的分化和成熟受到损害，并在异常T细胞(TC)反应的发展中发挥重要作用，从而导致疾病的发病。 背景和原理：DC是免疫系统的关键调节器和强大的TC刺激物。已有研究提出系统性自身免疫反应可通过DC功能的改变来解释。然而，关于SLE患者的DC是否存在异常分化、成熟以及与自体TC的异常相互作用，以及这些异常在SLE的发病中所起的作用，尚需进一步的研究。 具体目的：1.比较SLE患者和对照组单核细胞来源的DC的表型和功能参数2.确定SLE患者与对照组TC-DC相互作用的特异性，包括细胞增殖、分化/成熟和死亡。 方法： A)用流式细胞仪和多重RT-PCR方法检测SLE患者和健康对照组DC在特定刺激条件下的成熟和分化情况。用Annexin V/Propidium iodide染色比较两组DC对不同凋亡刺激的敏感性。 B)SLE和对照DC吞噬和内化凋亡和坏死细胞的能力将通过流式细胞仪和荧光显微镜下标记的凋亡或坏死细胞的DC掺入来评估。吞噬能力也将使用FITC-葡聚糖进行评估。 C)SLE和对照单核细胞来源的DC产生的细胞因子将采用多重ELISA法进行检测，结果经RT-PCR证实。 D)DC-TC的相互作用将通过使用流式细胞术、细胞毒性分析和与特定单抗的抑制分析来确定细胞类型和T细胞亚群的共培养中的凋亡敏感性来解决。 E)将通过流式细胞仪和H3掺入法评估自体DC在诱导SEE患者T细胞增殖和激活特定TC亚群方面的作用。 F)用流式细胞仪微珠阵列检测SLE DC在诱导TH1/TH2极化中的作用。 意义：这项资助中建议的研究结果可能确定SLE自身免疫反应和TC异常产生的潜在机制。此外，更好地了解SLE中DC分化和成熟的分子基础可能为调控DC成熟和/或DC向淋巴器官迁移提供概念性工具，从而限制对自身反应性T细胞的过度刺激。这些可能导致开发新的治疗干预措施，旨在逆转这些异常，并取消或阻止这种疾病的发病和/或严重程度。