Wiskott-Aldrich syndrome (WAS) is a disorder characterized by rare X-linked genetic immune deficiency with mutations in the Was gene, which is specifically expressed in hematopoietic cells. The spleen plays a major role in hematopoiesis and red blood cell clearance. However, to date, comprehensive analyses of the spleen in wild-type (WT) and WASp-deficient (WAS-KO) mice, especially at the transcriptome level, have not been reported. In this study, single-cell RNA sequencing (scRNA-seq) was adopted to identify various types of immune cells and investigate the mechanisms underlying immune deficiency. We identified 30 clusters and 10 major cell subtypes among 11,269 cells; these cell types included B cells, T cells, dendritic cells (DCs), natural killer (NK) cells, monocytes, macrophages, granulocytes, stem cells and erythrocytes. Moreover, we evaluated gene expression differences among cell subtypes, identified differentially expressed genes (DEGs), and performed enrichment analyses to identify the reasons for the dysfunction in these different cell populations in WAS. Furthermore, some key genes were identified based on a comparison of the DEGs in each cell type involved in specific and nonspecific immune responses, and further analysis showed that these key genes were previously undiscovered pathology-related genes in WAS-KO mice. In summary, we present a landscape of immune cells in the spleen of WAS-KO mice based on detailed data obtained at single-cell resolution. These unprecedented data revealed the transcriptional characteristics of specific and nonspecific immune cells, and the key genes were identified, laying a foundation for future studies of WAS, especially studies into novel and underexplored mechanisms that may improve gene therapies for WAS.

Wiskott-Aldrich 综合征 (WAS) 是一种罕见的 X 连锁遗传免疫缺陷性疾病，其特征是Was基因发生突变，该基因在造血细胞中特异性表达。脾脏在造血和红细胞清除中起着重要作用。然而，迄今为止，对野生型（WT）和WASp缺陷（WAS-KO）小鼠脾脏的综合分析，特别是在转录组水平上的分析尚未见报道。在本研究中，采用单细胞RNA测序（scRNA-seq）来识别各种类型的免疫细胞并研究免疫缺陷的机制。我们在 11,269 个细胞中鉴定出 30 个簇和 10 个主要细胞亚型；这些细胞类型包括 B 细胞、T 细胞、树突状细胞 (DC)、自然杀伤 (NK) 细胞、单核细胞、巨噬细胞、粒细胞、干细胞和红细胞。此外，我们评估了细胞亚型之间的基因表达差异，鉴定了差异表达基因（DEG），并进行了富集分析，以确定 WAS 中这些不同细胞群功能障碍的原因。此外，通过比较每种细胞类型中涉及特异性和非特异性免疫反应的DEG，鉴定了一些关键基因，进一步分析表明这些关键基因是WAS-KO小鼠中先前未发现的病理相关基因。总之，我们根据单细胞分辨率获得的详细数据展示了 WAS-KO 小鼠脾脏中免疫细胞的情况。这些前所未有的数据揭示了特异性和非特异性免疫细胞的转录特征，并确定了关键基因，为未来的WAS研究奠定了基础，特别是对可能改善WAS基因治疗的新颖且尚未探索的机制的研究奠定了基础。