Defects of Thymic Output in Wiskott-Aldrich Syndrome

Wiskott-Aldrich 综合征的胸腺输出缺陷

• 批准号: 7140254
• 负责人: EILEEN REMOLD-O'DONNELL
• 金额: $ 23.68万
• 依托单位: IMMUNE DISEASE INSTITUTE, INC.
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-07-01 至 2009-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7140254
• 关键词: T cell receptor; T lymphocyte; Wiskott Aldrich syndrome; cell population study; clinical research; developmental immunology; family genetics; human subject; immunopathology; pathologic process; patient oriented research; thymus disorder

DESCRIPTION (provided by applicant): The Wiskott-Aldrich syndrome (WAS) is a rare inherited platelet/ immunodeficiency disease characterized by thrombocytopenia, eczema, infections and frequency of autoimmune disorders and malignancies. The defective gene encodes WASP, a blood cell protein that coordinates cytoskeletal changes in endocytotic and proliferative responses of various blood cells. In a recent cross-sectional study of blood specimens, we found that WAS patients have decreased numbers of T and B lymphocytes. The deficit of cells was most pronounced for infant patients and for T cells primarily reflected a decrease of naive cells. Preliminary findings showed normal survival properties for naive T cells, suggesting that the cell deficit is due to deficient output. In the currently accepted paradigm for WAS, disease pathology is due to defective functions of peripheral blood cells. In the proposed R21 project, we will establish "proof of concept" for the novel hypothesis that defective thymocyte development and deficient output contribute significantly to the pathology of severe WAS. We will test for deficient thymic output by quantifying T cell receptor excision circles (TRECs) by real time PCR in CD4 and CDS cells of infant patients compared to normal infants. TREC values will be compared for WASP+ patients and WASP- patients, for whom frequency of autoimmune disease and malignancies is substantially higher. Measurement of the marker CD31 on CD4 naive cells (CD4+CD45RA+) will provide independent quantization of recent thymic emigrant cells. To eliminate other mechanisms as significant contributors to the cell deficit, patient naive CD4 and CD8 cells will be compared to normal cells for survival in vitro, levels of survival factors Bcl-2, Bcl-xL and IL7 receptor, and altered proliferation by analyzing nuclear antigen Ki-67 and ex vivo BrdU incorporation. Finally, we will determine whether naive cells of WAS patients have restricted/skewed antigen receptor repertoire. TCR repertoire diversity of sorted naive CD4 cells will be examined by CDR3 length analysis (spectratyping). Each T cell receptor (TCR) Vbeta fragment will be amplified with one of the 24 Vbeta-specific primers and a fluorescently labeled Cbeta. The results of the proposed 2 year proof-of-concept pilot project combined with our existing findings on mouse thymocytes would form the basis of a coordinated RO1 application combining study of patient T cells and WASP-null mouse models to delineate the role of WASP in thymocyte maturation.

描述（由申请人提供）：Wiskott-Aldrich综合征（WAS）是一种罕见的遗传性血小板/免疫缺陷疾病，其特征为血小板减少症、湿疹、感染以及自身免疫性疾病和恶性肿瘤的频率。缺陷基因编码WASP，一种血细胞蛋白，其协调各种血细胞的内吞和增殖反应中的细胞骨架变化。在最近的一项血液标本的横断面研究中，我们发现WAS患者的T和B淋巴细胞数量减少。对于婴儿患者，细胞的缺陷最明显，对于T细胞，主要反映了幼稚细胞的减少。初步研究结果表明，幼稚T细胞的正常存活特性，表明细胞缺陷是由于输出不足。在目前接受的WAS范例中，疾病病理是由于外周血细胞的功能缺陷。在拟议的R21项目中，我们将为新的假设建立“概念验证”，即胸腺细胞发育缺陷和输出不足对严重WAS的病理学有显著贡献。我们将通过真实的时间PCR定量婴儿患者与正常婴儿的CD 4和CDS细胞中的T细胞受体切除环（TRECs）来测试胸腺输出不足。将比较WASP+患者和WASP-患者的TREC值，其中自身免疫性疾病和恶性肿瘤的频率显著较高。对CD 4幼稚细胞（CD 4 + CD 45 RA+）上的标记物CD 31的测量将提供近期胸腺移出细胞的独立量化。为了消除作为细胞缺陷的重要贡献者的其他机制，将通过分析核抗原Ki-67和离体BrdU掺入来比较患者初始CD 4和CD 8细胞与正常细胞的体外存活、存活因子Bcl-2、Bcl-xL和IL 7受体的水平以及改变的增殖。最后，我们将确定WAS患者的幼稚细胞是否具有受限/偏斜的抗原受体库。将通过CDR 3长度分析（光谱分析）检查分选的初始CD 4细胞的TCR库多样性。每个T细胞受体（TCR）Vbeta片段将用24种Vbeta特异性引物之一和荧光标记的C β扩增。提出的2年概念验证试点项目的结果与我们现有的小鼠胸腺细胞研究结果相结合，将形成协调的RO 1应用的基础，结合患者T细胞和WASP缺失小鼠模型的研究，以描述WASP在胸腺细胞成熟中的作用。