Defects of Thymic Output in Wiskott-Aldrich Syndrome

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频率。该缺陷基因编码WASP，这是一种血细胞蛋白，在各种血细胞的内吞和增殖反应中协调细胞骨架的变化。在最近的一项血液标本横断面研究中，我们发现WAS患者的T淋巴细胞和B淋巴细胞数量减少。细胞缺陷在婴儿患者中最为明显，而T细胞主要反映了幼稚细胞的减少。初步结果显示幼稚T细胞的正常存活特性，表明细胞缺陷是由于输出不足。在目前被接受的WAS范式中，疾病病理是由于外周血细胞功能缺陷。在拟议的R21项目中，我们将为胸腺细胞发育缺陷和输出缺陷显著导致严重WAS病理的新假设建立“概念证明”。我们将通过实时PCR定量测定婴儿患者CD4和CDS细胞中的T细胞受体切除环（TRECs）来检测胸腺输出缺陷与正常婴儿的比较。将比较WASP+患者和WASP-患者的TREC值，WASP+患者自身免疫性疾病和恶性肿瘤的频率明显更高。测量CD4初始细胞（CD4+CD45RA+）上的CD31标记物将提供新近胸腺迁移细胞的独立量化。为了消除作为细胞缺陷重要因素的其他机制，将患者初始CD4和CD8细胞与正常细胞进行体外存活、生存因子Bcl-2、Bcl-xL和IL7受体水平的比较，并通过分析核抗原Ki-67和体外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有限制/扭曲的抗原受体库。通过CDR3长度分析（谱型分析）检测分类后初始CD4细胞的TCR库多样性。每个T细胞受体（TCR） Vbeta片段将用24种Vbeta特异性引物中的一种和荧光标记的Cbeta扩增。这个为期2年的概念验证试点项目的结果，结合我们在小鼠胸腺细胞上的现有发现，将构成一个协调的RO1应用的基础，结合患者T细胞和WASP-null小鼠模型的研究，来描述WASP在胸腺细胞成熟中的作用。

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.