WAS蛋白缺陷对记忆CD4+T细胞受体多样性的影响

T cell receptor (TCR) diversity is an important base of the body to achieve effective immune protection. For the first time, we found that the TCR diversity of memory CD4+T cells was significantly skewed in young WAS patients as compared with that in the age-matched healthy controls, but the underlying mechanisms remain unknown. Recurrent or chronic infections are generally considered the most common acquired factors influencing TCR diversity, however, clinical research in young WAS patients is difficult to prove this. As a result, we propose a scientific hypothesis: the T cell intrinsic defects caused by WAS protein (WASp) deficiency rather than recurrent infections is the root cause of the skewed TCR diversity in memory CD4+T cells in young WAS patients or mice. This study intends to establish a bone marrow chimeric model of the WAS knock-out mice (WKO, CD45.2 +) and wild type mice (WT, CD45.1 +), then analyze the TCR diversity in different memory CD4+T cell subsets in thymus and spleen of WKO and WT(CD45.2+). In addition, we will analyze the TCR diversity of different memory CD4+T cells in WASp+ and WASp- cells in young WAS back mutation patients. The validation of scientific hypothesis will not only lay a solid ground for further research on the mechanism of how WASp affect TCR diversity, it will also help to further clarify the complex function of WASp, and may provide new clues to optimize the design of the vaccine for WAS patients.

T细胞受体（TCR）多样性是机体实现有效免疫保护的重要基础。我们首次发现年幼WAS患儿记忆CD4+T细胞TCR多样性较正常同龄对照显著受限，但机制不清。反复或慢性感染通常被认为是影响TCR多样性最常见后天因素，年幼WAS病人的临床研究中却很难被证实。由此，我们提出科学假设：WASp缺陷导致的T细胞内在缺陷而非感染是CD4+T细胞TCR多样性受限的根本原因。本研究拟采用WAS基因敲除鼠（WKO，CD45.2+）与野生型小鼠（CD45.1+）骨髓嵌合模型，研究其胸腺和脾脏中WKO与野生型小鼠记忆CD4+T细胞TCR多样性；并以我们多年累积诊断的年幼WAS回复突变患儿作为研究对象，探讨其体内WASp+和WASp-细胞亚群记忆CD4TCR的特点。科学假设的验证不仅将为进一步研究WASp影响TCR的具体机制奠定基础，亦将助于进一步阐明WASp这一结构复杂蛋白质的功能，并可能为优化疫苗设计提供新线索。

T细胞受体(TCR)多样性是机体实现有效免疫保护的重要基础。我们既往的研究发现年幼WAS患儿记忆CD4+T细胞TCR多样性较正常同龄对照显著受限，但这一表型是否由WAS蛋白功能缺陷直接导致仍不清。反复或慢性感染通常被认为是影响TCR多样性最常见后天因素，在临床研究中很难避免外在因素的干扰。由此，我们采用WAS基因敲除鼠(WKO，CD45.2+)与野生型小鼠(WT，CD45.1+)骨髓嵌合模型，对比研究WKO与WT小鼠其脾脏中记忆CD4+T细胞、CD8+T细胞TCR多样性；同时，利用单细胞测序技术研究罕见的年幼WAS回复突变患儿体内WASp+和WASp-细胞亚群记忆CD4TCR的特点。动物实验结果发现：WASp缺陷可导致CD4+TEM的TCR多样性轻度受限，而不影响CD8+TCM的TCR多样性；WASp缺陷可导致CD4+TEM和CD8+TCM的TRB的V、D、J亚基因的取用，V、D、J亚基因组合，以及氨基酸水平发生变化；但WAS缺陷不影响TCR的疏水性及CDR3序列长度。该结果表明WASp缺陷本身可导致CD4+T细胞TCR多样性受限。回复突变患儿的数据还需进一步解析。本课题研究结果将为进一步研究WASp影响TCR的具体机制奠定基础，亦将助于进一步阐明WASp这一结构复杂蛋白质的功能，并可能为优化疫苗设计提供新线索。