PDF(Pubmed)
Abstract：
Loss of function mutations in Diaphanous related formin 1 (DIAPH1) are associated with seizures, cortical blindness, and microcephaly syndrome (SCBMS) and are recently linked to combined immunodeficiency. However, the extent of defects in T and innate lymphoid cells (ILCs) remain unexplored. Herein, we characterized the primary T, natural killer (NK) and helper ILCs of six patients carrying two novel loss of function mutation in DIAPH1 and Jurkat cells after DIAPH1 knockdown. Mutations were identified by whole exome sequencing. T-cell immunophenotyping, proliferation, migration, cytokine signaling, survival, and NK cell cytotoxicity were studied via flow cytometry-based assays, confocal microscopy, and real-time qPCR. CD4+ T cell proteome was analyzed by mass spectrometry. p.R351* and p.R322*variants led to a significant reduction in the DIAPH1 mRNA and protein levels. DIAPH1-deficient T cells showed proliferation, activation, as well as TCR-mediated signaling defects. DIAPH1-deficient PBMCs also displayed impaired transwell migration, defective STAT5 phosphorylation in response to IL-2, IL-7 and IL-15. In vitro generation/expansion of Treg cells from naïve T cells was significantly reduced. shRNA-mediated silencing of DIAPH1 in Jurkat cells reduced DIAPH1 protein level and inhibited T cell proliferation and IL-2/STAT5 axis. Additionally, NK cells from patients had diminished cytotoxic activity, function and IL-2/STAT5 axis. Lastly, DIAPH1-deficient patients\' peripheral blood contained dramatically reduced numbers of all helper ILC subsets. DIAPH1 deficiency results in major functional defects in T, NK cells and helper ILCs underlining the critical role of formin DIAPH1 in the biology of those cell subsets.
摘要：
透明相关形式1（DIAPH1）的功能缺失突变与癫痫发作有关，皮质失明,和小头畸形综合征（SCBMS），最近与联合免疫缺陷有关。然而,T细胞和先天淋巴细胞（ILC）的缺陷程度仍未被研究。在这里，我们描述了主要的T，在DIAPH1敲低后，6名患者在DIAPH1和Jurkat细胞中携带两种新型功能缺失突变。通过全外显子组测序鉴定突变。T细胞免疫分型,扩散,迁移,细胞因子信号，生存,和NK细胞的细胞毒性研究通过流式细胞术为基础的测定，共聚焦显微镜，和实时qPCR。通过质谱分析CD4+T细胞蛋白质组。p.R351*和p.R322*变体导致DIAPHlmRNA和蛋白质水平的显著降低。DIAPH1缺陷型T细胞表现出增殖,激活,以及TCR介导的信号缺陷。DIAPH1缺陷的PBMC也表现出受损的跨肠迁移，响应IL-2，IL-7和IL-15的STAT5磷酸化缺陷。来自初始T细胞的Treg细胞的体外生成/扩增显著减少。shRNA介导的Jurkat细胞中DIAPH1沉默降低DIAPH1蛋白水平并抑制T细胞增殖和IL-2/STAT5轴。此外,患者的NK细胞具有减弱的细胞毒活性,功能和IL-2/STAT5轴。最后,DIAPH1缺陷患者的外周血中所有辅助ILC亚群的数量显著减少。DIAPH1缺陷导致T的主要功能缺陷，NK细胞和辅助性ILC强调了形式素DIAPH1在这些细胞亚群生物学中的关键作用。
