FOXP3 ΔE2 Isoform in Treg Function and Pathophysiology of Lupus

项目总结 Foxp3是调节性T细胞(Tregs)发育和功能的关键转录因子 人类和小鼠该基因的非晶态突变导致全身性自身免疫性疾病和变态反应 症状。Foxp3基因高度保守，人和小鼠的氨基酸同源性为87%。人类 然而，Foxp3有两种主要的亚型，全长(FOXP3FL亚型)和选择性剪接亚型 缺乏外显子2(FOXP3FOXP3FL亚型)，而小鼠∆基因只产生FOXP3FL亚型。角色 这两种异构体在健康和疾病中的作用还没有明确的定义。在这份赠款中，我们提出了证据 由于突变而只表达FOXP3ΔE2亚型的患者会发生自身免疫性疾病。我们 建立FOXP3外显子2缺失小鼠模型，研究FOXP3ΔE2亚型在体内和体外的功能 证明FOXP3ΔE2小鼠产生了类似于系统性狼疮的系统性自身免疫 红斑狼疮(SLE)。我们进一步证明了只表达FOXP3ΔE2亚型的Treg是 不稳定，向效应器T辅助细胞转分化，并在以下情况下足以诱发SLE样疾病 转移到Tcrb-/-小鼠体内。我们推测，FOXP3ΔE2是人类两种主要亚型之一，是一种 系统性红斑狼疮发生和/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。(1) 明确FOXP3、Δ、E2、Tregs在狼疮发病和严重程度中的作用。我们假设FOXP3ΔE2树有一个 与Foxp3 FL Tregs不同的转录程序，使它们具有谱系不稳定性。在狼疮下 诱导环境中，FOXP3ΔE2树在外围失去谱系身份并转分化为 自身反应性效应细胞，即使在Foxp3存在的情况下也会导致更早的发病和/或更严重的疾病 FL Tregs.(2)明确FOXP3ΔE2亚型对人类Treg表型和病程的影响。 系统性红斑狼疮。我们假设FOXP3亚型表达的比例决定了人类Treg 系统性红斑狼疮的表型及其与病程的相关性。我们将确定FOXP3是否 ΔE2：FOXP3FL比率，主要表达ΔE2亚型的Tregs的存在和频率相关 用流式细胞术和专门设计的单细胞RNA-Seq方法检测SLE的病情。我们 将进一步测试SLE患者的细胞因子环境是否调节FOXP3前-mRNA剪接，从而改变 同构表达式。从这些研究中获得的信息将为生理学研究提供新的见解。 自然存在的人FOXP3ΔE2亚型的功能，并将为开发高效的 旨在改变FOXP3亚型表达作为治疗SLE及其他疾病的临床方案 自身免疫性疾病。

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.