Mechanism of Humoral Immune Defects in Autoimmune Polyglandular Syndrome Type 1

项目摘要 自身免疫性多腺体综合征1型(APS-1)是由自身免疫调节因子(AIRE)突变引起的 基因，它通常促进中枢和外周T细胞耐受。APS-1患者表现出自身免疫性疾病 并易患慢性皮肤粘膜念珠菌病(CMC)。缺乏机械性的-- 念珠菌病的基础及其与自身免疫的矛盾联系阻碍了有效的治疗 APS-1。念珠菌病患者往往存在细胞免疫缺陷。APS-1患者产生类- 抗辅助性T细胞17(TH17)细胞因子的转换型免疫球蛋白自身抗体，可中和和损害保护性抗 真菌免疫力。这突显了体液免疫功能障碍是导致CMC的潜在重要因素 APS-1。我们的长期目标是了解自身免疫和免疫缺陷疾病的免疫失调。 DES和开发机制导向的治疗策略。本申请的目的是阐明 APS-1的体液免疫缺陷机制。我们的初步研究表明，AIRE是专门表达的 人和小鼠生发中心(GC)B细胞在体内次级淋巴器官和在B细胞 体外抗体的多样化。B细胞AIRE缺陷导致类开关重组(CSR)升高和 体细胞超突变(SHM)。当AIRE被引入AIRE缺陷的B细胞时，AIRE抑制CSR。AIRE在 B细胞具有激活诱导胞苷脱氨酶(AID)，这是SHM和CSR所必需的酶。艾尔-/-老鼠有 全身和粘膜淋巴组织中GC B细胞和T细胞反应的异常扩张。这些发现 支持中心假设，即APS-1与B细胞引起的抗体成熟异常增加有关- AIRE介导的AID调节中的内在缺陷和自身反应选择中的B细胞外源性缺陷 由于GC反应过度而产生的抗体。我们的理论基础是体液免疫机制的阐明 APS-1的缺陷将促使对APS-1进行更具体的治疗。这一假设将通过追求两个具体的 目标。采用实时定量聚合酶链式反应、流式细胞术、染色质免疫沉淀、基因组尿嘧啶斑点杂交等方法 和下一代测序，目标1的研究将确定抗体成熟和 APS-1抗体库及AIRE调节B细胞AID的机制AIM 2中的研究将使用 竞争性酶联免疫吸附试验以及小鼠皮肤白色念珠菌感染模型，以确定B细胞- APS-1相关性念珠菌病的固有和非固有AIRE缺陷。这项研究提出了创新和削减-- EDGE方法学在APS-1发病机制中测试新的和机械的假设。这项工作将持续下去 以及对基础和临床免疫学领域的广泛影响，因为它不仅将促进对 疾病APS-1和AID介导的抗体成熟调节，但也提供了对AN之谜的洞察 与自身免疫共存的免疫缺陷的数量不断增加，从而为更好地治疗- 这是一种疾病。

Project Summary Autoimmune Polyglandular Syndrome type 1 (APS-1) is caused by mutations in the Autoimmune Regulator (Aire) gene, which normally promotes central and peripheral T cell tolerance. APS-1 patients exhibit autoimmune disorders in multiple organs and a predisposition to chronic mucocutaneous candidiasis (CMC). A lack of mechanistic under- standing of the basis of candidiasis and its paradoxical association with autoimmunity hampers effective treatment of APS-1. Patients with candidiasis often have defects in cell-mediated immunity. APS-1 patients produce class- switched IgG autoantibodies against T helper 17 (TH17) cytokines, which may neutralize and impair protective anti- fungal immunity. This highlights humoral immune dysfunction as a potentially important contributing factor to CMC in APS-1. Our long-term goal is to understand the immune dysregulation in autoimmune and immunodeficiency disor- ders and to develop mechanism-guided therapeutic strategies. The objective in this application is to elucidate the mechanism of humoral immune defects in APS-1. Our preliminary studies show that AIRE is expressed specifically in human and mouse germinal center (GC) B cells in secondary lymphoid organs in vivo, and in B cells undergoing antibody diversification in vitro. AIRE deficiency in B cells causes elevated class switch recombination (CSR) and somatic hypermutation (SHM). AIRE suppresses CSR when introduced into AIRE-deficient B cells. AIRE interacts in B cells with activation-induced cytidine deaminase (AID), the enzyme essential for SHM and CSR. Aire‒/‒ mice have aberrant expansion of GC B cell and T cell responses in systemic and mucosal lymphoid tissues. These findings support the central hypothesis that APS-1 involves aberrantly increased antibody maturation caused by B cell- intrinsic defects in AIRE-mediated AID regulation and B cell-extrinsic defects in the selection against autoreactive antibodies due to excessive GC response. Our rationale is that the elucidation of the mechanism of humoral immune defects in APS-1 will prompt more specific treatment of APS-1. The hypothesis will be tested by pursing two specific aims. Employing quantitative real-time PCR, flow cytometry, chromatin immunoprecipitation, genomic uracil dot blot and next-generation sequencing, studies in Aim 1 will determine the abnormalities in antibody maturation and the antibody repertoire in APS-1 and the mechanism by which AIRE regulates AID in B cells. Studies in Aim 2 will use competitive ELISA as well as mouse models of cutaneous Candida albicans infection to determine the role of B cell- intrinsic and -extrinsic AIRE deficiency in APS-1-associated candidiasis. This study proposes innovative and cutting- edge methodologies to test novel and mechanistic hypotheses in APS-1 pathogenesis. The work will have sustained and broad impact on the field of basic and clinical immunology, as it will not only advance the understanding of the disease APS-1 and AID-mediated regulation of antibody maturation, but also offer insights into the enigmas of an increasing number of immunodeficiencies that co-present with autoimmunity, thus paving the way to better treat the- se diseases.