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: 7073472
• 负责人: Mariana J Kaplan
• 金额: $ 7.47万
• 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 2008-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7073472
• 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（由申请人提供）：假设：在系统性红斑狼疮（SLE）中，髓样树突状细胞（My-DC）的分化和成熟受损，并在导致疾病发病机制的异常T细胞（TC）应答的发展中发挥重要作用。 背景和原理：DC是免疫系统的关键调节剂和有效的TC刺激剂。有人提出，系统性自身免疫反应的特点SLE可以解释DC功能的改变。然而，需要了解SLE患者的DC是否具有异常分化、成熟和与自体TC的异常相互作用，以及这些异常在SLE发病机制中可能发挥的作用。 具体目标：1.比较SLE患者和正常人单核细胞来源DC的表型和功能参数。确定与对照组相比，SLE患者中TC-DC相互作用的具体特征，包括细胞增殖，分化/成熟和死亡。 研究方法： A）将通过流式细胞术和多重RT-PCR在SLE患者和健康对照中评估响应于特异性刺激的DC成熟和分化。将在两组中用膜联蛋白V/碘化丙啶染色比较DC对不同凋亡刺激的敏感性。 B）使用流式细胞术和荧光显微镜通过CMFDA标记的凋亡或坏死细胞的DC掺入来评价SLE和对照DC吞噬和内化凋亡和坏死细胞的能力。还将使用FITC-葡聚糖评价吞噬能力。 C）使用多重ELISA测定SLE和对照单核细胞衍生的DC的细胞因子产生，并通过RT-PCR确认结果。 D）DC-TC相互作用将通过使用流式细胞术、细胞毒性测定和使用特异性单克隆抗体的抑制测定来确定细胞类型和T细胞亚群的共培养物中的细胞凋亡易感性来解决。 E）自体DC在SEE患者中诱导T细胞增殖和特异性TC亚群活化中的作用将通过流式细胞术和H3掺入来评估。 F）SLE DC在诱导TH 1/TH 2极化中的作用将通过细胞计数珠阵列来评估。 重要性：这项研究的结果可能会发现参与SLE自身免疫反应和TC异常的潜在机制。此外，更好地了解SLE中DC分化和成熟的分子基础可能提供概念工具来调节DC成熟和/或DC向淋巴器官的迁移，这可能限制自身反应性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，消除或阻止这种疾病的发作和/或严重程度。