FOXP3 ΔE2 Isoform in Treg Function and Pathophysiology of Lupus

FOXP3 αE2 亚型在 Treg 功能和狼疮病理生理学中的作用

• 批准号: 10391526
• 负责人: BAOHUA ZHOU
• 金额: $ 19.81万
• 依托单位: INDIANA UNIV-PURDUE UNIV AT INDIANAPOLIS
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-04-12 至 2024-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10391526
• 关键词: Alleles; Allergic; Allergic Disease; Alternative Splicing; Amino Acids; Asthma; Autoimmune Diseases; CD4 Positive T Lymphocytes; Cells; Clinical Protocols; Data; Deletion Mutation; Development; Disease; Ectopic Expression; Effector Cell; Environment; Exons; FOXP3 gene; Female; Flow Cytometry; Frequencies; Functional disorder; Grant; Health; Homeostasis; Human; Hypersensitivity; Immune; Immune Tolerance; Immune response; Immune system; In Vitro; Inflammation; Lead; Length; Link; Lupus; Mediating; Methods; Modeling; Mus; Mutation; Outcome; Patients; Pharmacology; Phenotype; Physiological; Play; Protein Isoforms; RNA Splicing; Regulatory Element; Regulatory T-Lymphocyte; Risk; Risk Factors; Role; Severities; Symptoms; Syndrome; Systemic Lupus Erythematosus; Testing; Therapeutic; Thymus Gland; X Inactivation; autoreactivity; base; cytokine; demethylation; design; early onset; in vivo; insight; mRNA Precursor; novel; novel strategies; overexpression; programs; single-cell RNA sequencing; systemic autoimmune disease; systemic autoimmunity; transcription factor; transcriptomics; treatment strategy

PROJECT SUMMARY FOXP3 is a key transcription factor for the development and function of regulatory T cells (Tregs) and amorphic mutations at this locus in humans and mice lead to systemic autoimmune diseases and allergic symptoms. FOXP3 gene is highly conserved with 87% amino acid identity between humans and mice. Human FOXP3 however has two major isoforms, a full length (FOXP3 FL isoform) and an alternatively spliced isoform lacking exon 2 (FOXP3 ∆E2 isoform), while mouse Foxp3 gene only produce the FOXP3 FL isoform. The roles of these two isoform in health and disease have not been clearly defined. In this grant we present evidence that patients who only express the FOXP3 ΔE2 isoform due to mutations developed autoimmune diseases. We generated mice with Foxp3 exon 2 deletion to study the functionality of FOXP3 ΔE2 isoform in vivo and demonstrated that Foxp3 ΔE2 mice developed a systemic autoimmunity resembling systemic lupus erythematosus (SLE). We further demonstrated that Tregs expressing only the Foxp3 ΔE2 isoform were unstable, transdifferentiated to effector T helpers, and were sufficient to induce SLE-like disease when transferred into Tcrb-/- mice. We hypothesize that FOXP3 ΔE2, one of the two major isoforms in humans, is a risk factor for development and/or severity of SLE. We will test our hypothesis in the following specific aims. (1) Define the role of Foxp3 ΔE2 Tregs on lupus onset and severity. We hypothesize that Foxp3 ΔE2 Tregs have a different transcriptomic program from Foxp3 FL Tregs and render them to be lineage instability. Under lupus inducing environment, Foxp3 ΔE2 Tregs lose lineage identity in the periphery and transdifferentiate into autoreactive effector cells, resulting earlier onset and/or more severe disease even in the presence of Foxp3 FL Tregs. (2) Define the impact of FOXP3 ΔE2 isoform on human Treg phenotypes and disease course of systemic lupus. We hypothesize that the ratio of FOXP3 isoform expression determines human Treg phenotypes and correlates with disease course of systemic lupus. We will determine whether the FOXP3 ΔE2:FOXP3 FL ratio, the existence and frequency of Tregs expressing mainly FOXP3 ΔE2 isoform correlate with disease state of SLE using flow cytometry and a specially designed method of single cell RNA-Seq. We will further test whether cytokine milieu in SLE patients regulates FOXP3 pre-mRNA splicing thus shift the isoform expression. The information learned from these studies will provide novel insights into the physiological functions of the naturally existing human FOXP3 ΔE2 isoform and would pave the way to develop efficient clinical protocols aimed at shifting FOXP3 isoform expression as therapeutic treatment of SLE as well as other autoimmune diseases.

项目摘要 FOXP 3是调节性T细胞（TCFs）发育和功能的关键转录因子， 在人类和小鼠中该基因座的无定形突变导致全身性自身免疫疾病和过敏性疾病。 症状FOXP 3基因在人和小鼠之间高度保守，氨基酸同源性为87%。人类 然而，FOXP 3有两种主要的同种型，全长（FOXP 3 FL同种型）和选择性剪接同种型 缺乏外显子2（FOXP 3 E2同种型），而小鼠Foxp 3基因仅产生FOXP 3 FL同种型。的角色 这两种亚型在健康和疾病中的作用还没有明确的定义。在这份授权书中，我们提出了证据， 由于突变而仅表达FOXP 3 ΔE2亚型的患者发展为自身免疫性疾病。我们 产生Foxp 3外显子2缺失的小鼠以研究体内FOXP 3 ΔE2同种型的功能性， 表明Foxp 3 ΔE2小鼠产生了类似系统性狼疮的系统性自身免疫， 红斑性狼疮（SLE）。我们进一步证明了仅表达Foxp 3 ΔE2亚型的TcR是一种高表达的细胞。 不稳定，转分化为效应T辅助细胞，并且当 转移到Tcrb-/-小鼠中。我们假设FOXP 3 ΔE2（人类两种主要亚型之一）是一种 SLE发展和/或严重程度的危险因素。我们将在以下具体目标中检验我们的假设。（一） 定义Foxp 3 ΔE2 T在狼疮发作和严重程度中的作用。我们假设Foxp 3 ΔE2 T β具有一个 不同的转录组程序从Foxp 3 FL Tclase，并使它们成为谱系不稳定性。狼疮下 在诱导环境中，Foxp 3 ΔE2 T细胞在外周失去谱系同一性，并转分化为 自身反应性效应细胞，即使在Foxp 3存在下也导致更早发病和/或更严重的疾病 佛罗里达州Tennis。(2)确定FOXP 3 ΔE2亚型对人类Treg表型和疾病进程的影响， 系统性狼疮我们假设FOXP 3亚型表达的比例决定了人类Treg 系统性狼疮的表型与病程相关。我们将确定FOXP 3 ΔE2：FOXP 3 FL比值、主要表达FOXP 3 ΔE2亚型的TcB的存在和频率与 采用流式细胞术和专门设计的单细胞RNA-Seq方法，我们 将进一步测试SLE患者中的细胞因子环境是否调节FOXP 3前mRNA剪接，从而改变细胞因子的表达。 同种型表达。从这些研究中获得的信息将为生理学提供新的见解。 天然存在的人类FOXP 3 ΔE2亚型的功能，并将为开发有效的 旨在改变FOXP 3同种型表达作为SLE以及其他疾病的治疗性治疗的临床方案 自身免疫性疾病