Defects of Thymic Output in Wiskott-Aldrich Syndrome

Wiskott-Aldrich 综合征的胸腺输出缺陷

• 批准号: 6957407
• 负责人: EILEEN REMOLD-O'DONNELL
• 金额: $ 28.41万
• 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 2007-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6957407
• 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，其特征是血小板减少、湿疹、感染以及自身免疫性疾病和恶性肿瘤的发生率。该缺陷基因编码 WASP，一种血细胞蛋白，可协调各种血细胞的内吞和增殖反应中的细胞骨架变化。在最近的一项血液样本横断面研究中，我们发现 WAS 患者的 T 和 B 淋巴细胞数量减少。婴儿患者的细胞缺陷最为明显，而 T 细胞的缺陷主要反映了幼稚细胞的减少。初步结果显示初始 T 细胞具有正常的存活特性，表明细胞缺陷是由于输出不足造成的。在目前公认的WAS范式中，疾病病理学是由于外周血细胞的功能缺陷造成的。在拟议的 R21 项目中，我们将为新假设建立“概念验证”，即胸腺细胞发育缺陷和输出不足对严重 WAS 的病理学有显着影响。我们将通过实时 PCR 定量婴儿患者与正常婴儿的 CD4 和 CDS 细胞中的 T 细胞受体切除环 (TREC)，来测试胸腺输出不足。将比较 WASP+ 患者和 WASP- 患者的 TREC 值，这些患者自身免疫性疾病和恶性肿瘤的发生率要高得多。 CD4 幼稚细胞 (CD4+CD45RA+) 上标记 CD31 的测量将提供最近胸腺移出细胞的独立量化。为了消除导致细胞缺陷的其他重要机制，将患者初始 CD4 和 CD8 细胞与正常细胞进行体外存活、存活因子 Bcl-2、Bcl-xL 和 IL7 受体水平的比较，并通过分析核抗原 Ki-67 和离体 BrdU 掺入来改变增殖。最后，我们将确定 WAS 患者的初始细胞是否具有受限/倾斜的抗原受体库。将通过 CDR3 长度分析（光谱分型）检查分选的初始 CD4 细胞的 TCR 库多样性。每个 T 细胞受体 (TCR) Vbeta 片段将使用 24 个 Vbeta 特异性引物之一和荧光标记的 Cbeta 进行扩增。拟议的为期 2 年的概念验证试点项目的结果与我们对小鼠胸腺细胞的现有发现相结合，将构成协调 RO1 应用的基础，该应用结合了患者 T 细胞和 WASP 无效小鼠模型的研究，以描述 WASP 在胸腺细胞成熟中的作用。