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.

项目摘要 IgA肾病是世界上最常见的原发性肾小球肾炎，30%-50%的患者 进展为终末期肾病。诊断是以活检为基础的，采用常规的免疫荧光 表现为以免疫球蛋白A(共)为主的免疫沉积，通常伴有C3，常与免疫球蛋白共沉积。免疫球蛋白A在 免疫沉淀物属于IgA1亚类，富含缺乏半乳糖的IgA1糖型(Gd-IgA1)。的确有 由于我们对其潜在机制的了解有限，目前还没有针对IgAN的疾病特异性治疗方法。 疾病的进展。最近的数据表明，几乎所有IgA肾病的免疫沉淀物中都存在免疫球蛋白G。 而且它富含Gd-IgA1特异性自身抗体。此外，Gd-IgA1和抗Gd-IgA1 IgA肾病患者循环中的免疫球蛋白自身抗体升高，其水平可预测疾病的进展。 基于这些和其他数据，我们提出了一种解释自身免疫本质的多重命中假说。 IgAN患者循环中Gd-Ig A1升高并被抗Gd-Ig A1抗体识别 自身抗体，导致循环免疫复合体的形成，其中一些沉积在 肾小球，刺激肾脏损伤。自身抗原的起源和特定细胞群的特征(S) 产生Gd-IgA1仍不清楚。最近，我们发现了从IgAN中永生化的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，只有 这些IgAN来源的细胞在细胞因子刺激(如IL-6)的刺激下增加Gd-IgA1的产生。关注 UP分析发现，STAT3和STAT1的激活仅在IgAN B细胞中增强，这是 细胞因子刺激后Gd-IgA1的产生。细胞因子刺激B细胞的单细胞转录组分析 IgAN患者细胞中发现IgHA1亚群相关基因异常表达 多种细胞因子信号通路的调控。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。 仅凭转录数据并不能确定Gd-IgA1的生产者。为了解决这个问题，我们开发了一部小说 凝集素与可靶向细胞表面的重组抗Gd-IgA1抗体的糖分型方法 Gd-IgA1的介绍。这一过程丰富了Gd-IgA1产量低和高的亚群，这些亚群具有 细胞因子刺激前后转录因子的差异激活。使用这个 用核苷酸连接的糖分型方法，可以靶向Gd-IgA1产生细胞进行转录组 分析、细胞内信号染色、染色质重塑和特异性自身抗原生成率。 总之，这些研究将阐明特定亚群中自身抗原产生的机制。 这可能会导致针对IgA肾病的新疾病疗法的开发。