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，其特征在于血小板减少，湿疹，自身免疫性疾病和恶性肿瘤的频率。缺陷的基因编码WASP，这是一种血细胞蛋白，可协调各种血细胞的内吞和增生反应的细胞骨架变化。在最近对血液标本的横断面研究中，我们发现患者的T和B淋巴细胞数量减少。对于婴儿患者而言，细胞的不足最为明显，而T细胞主要反映了幼稚细胞的降低。初步发现显示幼稚T细胞的正常存活率，表明细胞不足是由于输出不足引起的。在当前接受的wAS范式中，疾病病理是由于外周血细胞的功能缺陷所致。在拟议的R21项目中，我们将为新的假设建立“概念证明”，即胸腺细胞有缺陷的发育和不足产量对严重的病理产生了重大贡献。我们将通过与正常婴儿相比，通过实时PCR量化T细胞受体切除循环（TREC），通过实时PCR来测试胸腺输出不足。 WASP+患者和WASP患者将进行TREC值，因为它们的自身免疫性疾病的频率和恶性肿瘤的频率要高得多。标记CD31在CD4幼稚细胞（CD4+CD45RA+）上的测量将对最近的胸腺移民细胞独立量化。为了消除其他机制作为细胞不足的重要因素，将将患者幼稚的CD4和CD8细胞与正常细胞进行体外生存，生存因子水平Bcl-2，Bcl-XL和IL7受体的水平进行比较，并通过分析核抗原Ki-67和Ex vivo Brdu融合来改变增殖。最后，我们将确定患者的幼稚细胞是否受到限制/偏斜的抗原受体库。分类的幼稚CD4细胞的TCR曲目多样性将通过CDR3长度分析（SpectRatatyping）检查。每个T细胞受体（TCR）VBETA片段将用24个VBETA特异性引物之一和荧光标记的CBETA放大。拟议的2年概念验证试验项目的结果与我们在小鼠胸腺细胞上的现有发现相结合，将构成协调的RO1应用，结合了患者T细胞的研究和Wasp-Null小鼠模型，以描述黄蜂在胸腺细胞成熟中的作用。