Defects of Thymic Output in Wiskott-Aldrich Syndrome

描述(申请人提供)：Wiskott-Aldrich综合征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繁发生。该缺陷基因编码WASP，一种血细胞蛋白，协调各种血细胞的内吞和增殖反应中细胞骨架的变化。在最近的一项血液样本横断面研究中，我们发现IS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，T细胞的缺陷主要反映了幼稚细胞的减少。初步研究结果显示，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所致。在目前公认的WAS范式中，疾病病理学是由于外周血细胞功能缺陷造成的。在建议的R21项目中，我们将为胸腺细胞发育缺陷和输出不足对重症AS的病理有重要贡献的新假说建立“概念证明”。我们将通过实时定量聚合酶链式反应对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(TRECs)进行量化来检测胸腺输出缺陷。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。对CD_4原始细胞(CD_4、CD_4、CD_(45)RA)标志物CD31的测定将提供近期胸腺迁徙细胞的独立量化。为了消除导致细胞缺陷的其他重要机制，我们将通过分析核抗原Ki-67和体外BrdU掺入，比较患者原始CD4和CD8细胞与正常细胞在体外的存活情况、存活因子Bcl2、Bclxl和IL7受体的水平，并通过分析核抗原Ki-67和体外BrdU掺入改变其增殖。最后，我们将确定AS患者的原始细胞是否有限制/扭曲的抗原受体谱系。将通过CDR3长度分析(分型)检查分选的初始CD4细胞的TCR谱系多样性。每个T细胞受体(TCR)Vbeta片段将用24个Vbeta特异性引物之一和一个荧光标记的Cbeta进行扩增。拟议的为期两年的概念验证试点项目的结果与我们在小鼠胸腺细胞上的现有发现相结合，将形成一个协调的RO1应用程序，结合对患者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。

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.