Distinct Glycophenotypes with Abnormal Signaling Define a Subpopulation of B cells Responsible for Production of Galactose-Deficient IgA1, the Main Autoantigen in IgA Nephropathy

具有异常信号传导的独特糖表型定义了负责产生半乳糖缺陷型 IgA1（IgA 肾病的主要自身抗原）的 B 细胞亚群

• 批准号: 10563618
• 负责人: Colin Robert Reily
• 金额: $ 49.06万
• 依托单位: UNIVERSITY OF ALABAMA AT BIRMINGHAM
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-02-15 至 2027-11-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10563618
• 关键词: ATAC-seq; Acute; Address; Affect; Alleles; Antigen-Antibody Complex; Autoantibodies; Autoantigens; Autoimmune; Autoimmune Diseases; B-Lymphocytes; Binding; Biochemical; Biopsy; Blood; Cell secretion; Cell surface; Cells; Characteristics; Circulation; Clinical; Cytokine Signaling; Data; Deposition; Development; Diagnosis; Disease; Disease Progression; End stage renal failure; Enzymes; Exhibits; Flow Cytometry; Galactose; Gene Expression; Genes; Genetic Transcription; Genotype; Glomerulonephritis; Glycosyltransferase Gene; Hematuria; IGA Glomerulonephritis; IgA1; IgG autoantibodies; Immune; Immunofluorescence Immunologic; Immunoglobulin A; Immunoglobulin G; In Vitro; Inflammation Mediators; Injury; Injury to Kidney; Interleukin-10; Interleukin-4; Interleukin-6; Kidney; Lectin; Mediating; Methods; Nature; Nucleotides; Pathogenesis; Pathogenicity; Pathway interactions; Patients; Peripheral Blood Mononuclear Cell; Phenotype; Population; Process; Production; Prognosis; Protein Kinase; RNA analysis; Recombinants; Recurrence; Regulation; Role; STAT1 gene; STAT3 gene; Sampling; Serum; Sialyltransferases; Signal Induction; Signal Pathway; Signal Transduction; Single Nucleotide Polymorphism; Stains; TNFSF5 gene; Technology; Testing; Time; Transcriptional Activation; cell type; chromatin immunoprecipitation; chromatin remodeling; cofactor; cytokine; disease phenotype; follow-up; glycosylation; glycosyltransferase; inflammatory milieu; inhibitor; knock-down; novel; patient stratification; pharmacologic; response; transcription factor; transcriptome; transcriptomics; virtual

Project Summary IgA nephropathy (IgAN) is the most common primary glomerulonephritis in the world, with 30-50% of patients progressing to end-stage kidney disease. Diagnosis is biopsy-based, with routine immunofluorescence showing IgA (co)dominant immunodeposits usually with C3 and often with IgG co-deposits. IgA in the immunodeposits is of IgA1 subclass and enriched for galactose-deficient IgA1 glycoforms (Gd-IgA1). There is currently no disease specific therapy for IgAN due to our limited understanding of the underlying mechanisms of disease progression. Recent data indicate that IgG is present in immunodeposits of virtually all IgAN patients and that it is enriched for Gd-IgA1-specific autoantibodies. Furthermore, Gd-IgA1 and anti-Gd-IgA1 IgG autoantibodies are elevated in the circulation of IgAN patients and their levels predict disease progression. Based on these, and other data, we have proposed a multi-hit hypothesis explaining the autoimmune nature of IgAN: Gd-IgA1 is elevated in circulation of IgAN patients and is recognized by anti-Gd-IgA1 IgG autoantibodies, resulting in the formation of circulating immune complexes, some of which deposit in the glomeruli, inciting renal injury. The origin of the autoantigen and the characteristics of specific cell population(s) producing Gd-IgA1 remain unknown. Recently, we showed that immortalized IgA1-secreting cells from IgAN patients produce more Gd-IgA1 autoantigen compared to the cells from healthy controls. Furthermore, only these IgAN-derived cells increase Gd-IgA1 production in response to cytokine stimulation (e.g., IL-6). Follow up analysis found enhanced STAT3 and STAT1 activation only in IgAN B cells, and that it was necessary for Gd-IgA1 production following cytokine stimulation. Single-cell transcriptome analysis of cytokine stimulated B cells from IgAN patients found IgHA1-subpopulations with abnormal expression of genes responsible for regulation of multiple cytokine signaling pathways. These IgHA1-subpopulations also exhibited abnormal alterations in the expression level of glycosyltransferase enzymes relevant to IgA1 glycosylation. Transcriptional data alone does not identify Gd-IgA1 producers. To address this, we developed a novel glycophenotyping method using lectins and recombinant anti-Gd-IgA1 IgG that can target cell surface presentation of Gd-IgA1. This process enriched for low and high Gd-IgA1-producing subpopulations, which had differential activation of transcriptional factors before and after cytokine stimulation. Using this glycophenotyping method with nucleotide conjugation, we can target Gd-IgA1-producing cells for transcriptome analysis, intracellular signaling staining, chromatin remodeling, and specific autoantigen production rates. Together, these studies will elucidate the mechanisms of autoantigen production within specific subpopulations of IgA1-producing cells and potentially lead to the development of new disease therapies for IgAN.

项目概要 IgA 肾病 (IgAN) 是世界上最常见的原发性肾小球肾炎，约占患者的 30-50% 进展为终末期肾病。诊断以活检为基础，并进行常规免疫荧光检查 显示 IgA（共）显性免疫沉积，通常与 C3 共沉积，并且经常与 IgG 共沉积。 IgA 在 immunodeposits 属于 IgA1 亚类，富含半乳糖缺陷型 IgA1 糖型 (Gd-IgA1)。有 由于我们对潜在机制的了解有限，目前尚无针对 IgAN 的疾病特异性治疗方法 疾病进展。最近的数据表明，几乎所有 IgAN 的免疫沉积物中都存在 IgG 患者，并且它富含 Gd-IgA1 特异性自身抗体。此外，Gd-IgA1 和抗 Gd-IgA1 IgAN 患者循环中的 IgG 自身抗体升高，其水平可预测疾病进展。 基于这些和其他数据，我们提出了一个多重打击假设，解释了自身免疫的本质 IgAN：IgAN 患者循环中的 Gd-IgA1 升高，并被抗 Gd-IgA1 IgG 识别 自身抗体，导致循环免疫复合物的形成，其中一些沉积在 肾小球，诱发肾损伤。自身抗原的起源和特定细胞群的特征 产生 Gd-IgA1 的情况仍不清楚。最近，我们发现来自 IgAN 的永生化 IgA1 分泌细胞 与健康对照组的细胞相比，患者产生更多的 Gd-IgA1 自身抗原。此外，仅 这些 IgAN 衍生细胞响应细胞因子刺激（例如 IL-6）而增加 Gd-IgA1 的产生。跟随 up 分析发现 STAT3 和 STAT1 激活仅在 IgAN B 细胞中增强，并且这是必要的 细胞因子刺激后 Gd-IgA1 的产生。细胞因子刺激的 B 的单细胞转录组分析 来自 IgAN 患者的细胞发现 IgHA1 亚群的基因表达异常，这些基因负责 多种细胞因子信号通路的调节。这些 IgHA1 亚群也表现出异常 与 IgA1 糖基化相关的糖基转移酶表达水平的变化。 转录数据本身并不能识别 Gd-IgA1 生产者。为了解决这个问题，我们开发了一本小说 使用凝集素和可靶向细胞表面的重组抗 Gd-IgA1 IgG 的糖表型分析方法 Gd-IgA1 的呈现。该过程富集了低和高 Gd-IgA1 产生亚群，其中 细胞因子刺激前后转录因子的激活差异。使用这个 通过核苷酸缀合的糖表型分析方法，我们可以针对产生 Gd-IgA1 的细胞进行转录组分析 分析、细胞内信号染色、染色质重塑和特异性自身抗原产生率。 这些研究将共同​​阐明特定亚群内自身抗原产生的机制 产生 IgA1 的细胞，并有可能导致 IgAN 新疾病疗法的开发。