Detection of IL35+ Exosomes as a Marker for Peripheral Tolerance

检测 IL35 外泌体作为外周耐受性的标志物

• 批准号: 9978316
• 负责人: Jeremy A Sullivan
• 金额: $ 7.75万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-01-01 至 2021-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9978316
• 关键词: Address; Animals; Antibodies; Antigens; Ascites; Autoantigens; Autoimmune Process; Biological Assay; Blood; CD81 gene; Cell Culture Techniques; Cells; Control Animal; Culture Media; Data; Delayed Hypersensitivity; Detection; Effector Cell; Engineering; Enzyme-Linked Immunosorbent Assay; Failure; Family; Future; Hour; Human; Human Herpesvirus 4; Immune; Immune response; Immunize; Immunology; Immunosuppression; Inflammatory Response; Interleukin-12; Interleukins; Knockout Mice; Label; Lead; Liquid substance; LoxP-flanked allele; Lymph; Lymphocyte; Malignant Neoplasms; Measures; Mediating; Membrane; Mus; Physiological; Production; Property; Protein Subunits; Proteins; Protocols documentation; Regulation; Regulatory T-Lymphocyte; Reporter; Reporting; SCID Mice; Serum; Swelling; TNFSF5 gene; Testing; Tetanus Toxoid; Therapeutic; Transfusion; Transmission Electron Microscopy; Transplant Recipients; Transplantation; cell transformation; cytokine; design; exosome; extracellular vesicles; member; mouse model; neutralizing antibody; nonhuman primate; peripheral tolerance; response; transplant model

ABSTRACT Interleukin 35 (IL35) has emerged as a potent immuno-suppressive cytokine in cancer, auto-immune and transplant immunology. A member of the IL12 family of cytokines, IL35 is a heterodimeric cytokine composed of the protein subunits Epstein Barr Virus Induced 3 (Ebi3) and p35 and is produced and secreted predominantly by regulatory T cells (Tregs). While IL35 has been implicated in the regulation of a number of cellular immune responses, there still exists significant debate as to whether the cytokine actually exists. This debate is predicated on the fact that attempts to isolate IL35 from blood, physiological solutions like ascites or lymph, or even cell culture supernatants have failed. We have recently immune-precipitated both Ebi3 and p35 with an antibody to the tetraspanin CD81 from mouse lymphocytes. Tetraspanins are membrane spanning proteins associated with the formation of the small, extracellular vesicles, exosomes. This interesting observation prompted us to examine whether IL35 exists as Ebi3 and p35 proteins associated with tetraspanins and secreted and acquired as an exosome. To test the idea that IL35 exists as an exosome dependent cytokine, we designed the following specific aims: Specific Aim 1: To test whether exosomes isolated from serum of tolerized groups of tetraspanin knockout mice (CD81, CD63, CD9) lose Ebi3 and p35 positive exosomes. Specific Aim 2: To examine whether lymphocytes from tolerized-CD81, CD63 or CD9 knockout mice produce and release functional IL35+ immuno-suppressive exosomes. The successful completion of the outlined specific aims in this proposal will fill a significant gap in our understanding of the cytokine IL35, and will ideally lead to future applications that further our understanding of the therapeutic implications of IL35 for humans.

摘要 白介素35(IL35)在癌症、自身免疫和肿瘤等疾病中是一种强有力的免疫抑制细胞因子。 移植免疫学。IL35是细胞因子IL12家族中的一员，是一种异源二聚体细胞因子 爱泼斯坦-巴尔病毒诱导3(Ebi3)和p35蛋白亚基的产生和分泌 主要是由调节性T细胞(Treg)。虽然IL35被牵连到多项监管中 在细胞免疫反应方面，对于细胞因子是否确实存在仍存在重大争论。这 争论的基础是，试图从血液、腹水或其他生理溶液中分离出IL35 淋巴，甚至细胞培养上清液都失败了。我们最近已经免疫沉淀了ebi3和p35 用小鼠淋巴细胞中的Tetraspanin CD81抗体。Tetraspanins是跨膜的 与细胞外小泡、外体的形成有关的蛋白质。这很有趣 观察促使我们检查IL35是否以Ebi3和p35蛋白的形式存在 并以外体的形式分泌和获得。为了验证IL35作为外显体存在的想法 依赖细胞因子，我们设计了以下特定目标：特定目标1：检测外切体 从耐受组的Tetraspanin基因敲除小鼠(CD81、CD63、CD9)的血清中分离的Ebi3和p35丢失 阳性外显子。特异性目标2：检测免疫耐受的CD81、CD63或CD9的淋巴细胞 基因敲除小鼠产生并释放具有功能的IL35+免疫抑制外切体。成功者 完成这项建议中概述的具体目标将填补我们对 细胞因子IL35，并将理想地导致未来的应用，进一步加深我们对治疗性 IL35对人类的影响。