A single-cell atlas of immunocyte in spleen of a mouse model of Wiskott-Aldrich Syndrome

Wiskott-Aldrich syndrome (WAS) is a disorder characterized by rare X-linked genetic immune deficiency with mutations in the was gene, a gene expressed specifically in hematopoietic cells, and the spleen plays a major role in hematopoiesis and the clearance of red blood cell. However, till now, comprehensive analyses of the spleen between wild type (WT) and WASp-deficient (WAS-KO) mice, especially at the transcriptomic level, have not been studied. Here, single-cell RNA sequencing (scRNA-seq) was adopted to identify various types of immune cells and investigate the mechanism of immune deficiency. We identified 30 clusters and 10 major cell subtypes among 11, 269 cells, including B cell, T cell, dendritic cell (DC), Natural Killer (NK) cell, monocyte, macrophage, granulocyte, stem cell and erythrocyte. Meanwhile, we evaluated the gene expression differences of cell subtypes, and analyzed the differential gene expression (DEGs) and enrichment analyses to reveal the reasons for the dysfunction of these different cell populations in WAS. Furthermore, some key genes were screened out by comparing the DEGs of each cell type during specific and non-specific immunization, and further analysis showed that these key genes were newly discovered pathologically related genes in WAS-KO mice. In summary, we present a detailed single cell resolution landscape of immune cells in spleen of WAS-KO mice. These unprecedented data uncovered the transcriptional characteristics of specific and non-specific immune cells, and the key genes were identified to lay a foundation for future studies of WAS, especially in discovering novel and underexplored mechanisms to improve gene therapies for WAS. Single cells were encapsulated in droplets using 10 x Genomics GemCode Technology and processed according to the manufacturer's instruction. scRNA-seq was adopted to identify various types of immune cells and investigate the mechanism of immune deficiency.维斯科特-奥尔德里奇综合征（Wiskott-Aldrich syndrome, WAS）是一类罕见的X连锁遗传性免疫缺陷病，由was基因突变所致；该基因仅在造血细胞中特异性表达，而脾脏在造血功能与红细胞清除过程中发挥核心调控作用。然而迄今为止，针对野生型（wild type, WT）与WAS蛋白缺陷型（WASp-deficient, WAS-KO）小鼠脾脏的全面系统分析，尤其是转录组层面的研究，尚未见相关报道。 本研究采用单细胞RNA测序（single-cell RNA sequencing, scRNA-seq）技术，对各类免疫细胞进行鉴定并探究免疫缺陷的发病机制。我们在11269个细胞中鉴定出30个细胞簇与10种主要细胞亚型，涵盖B细胞、T细胞、树突状细胞（dendritic cell, DC）、自然杀伤（Natural Killer, NK）细胞、单核细胞、巨噬细胞、粒细胞、干细胞及红细胞。同时，我们评估了各细胞亚型的基因表达差异，通过差异基因表达（differential gene expression, DEGs）分析与功能富集分析，揭示了WAS模型中不同细胞群体功能异常的潜在分子机制。此外，通过对比特异性与非特异性免疫刺激下各细胞类型的差异表达基因，我们筛选出若干关键基因；进一步分析显示，这些关键基因是WAS-KO小鼠中全新发现的病理相关基因。 综上，本研究构建了WAS-KO小鼠脾脏免疫细胞的高精度单细胞分辨率转录图谱。这些前所未有的数据揭示了特异性与非特异性免疫细胞的转录特征，所鉴定的关键基因为未来WAS的研究奠定了坚实基础，尤其为探索未被充分阐明的全新机制以优化WAS基因治疗方案提供了重要依据。 本研究采用10× Genomics GemCode技术将单细胞包裹于微滴中，并严格遵循制造商的操作指南完成后续实验流程。单细胞RNA测序（scRNA-seq）技术被用于鉴定各类免疫细胞并探究免疫缺陷的发病机制。