Detection of IL35+ Exosomes as a Marker for Peripheral Tolerance

摘要 白细胞介素35（IL 35）已经成为癌症、自身免疫和免疫性疾病中有效的免疫抑制细胞因子。 移植免疫学作为细胞因子的IL 12家族的成员，IL 35是异二聚体细胞因子，其由以下组成： 蛋白亚基Epstein巴尔病毒诱导3（Ebi 3）和p35的结合，并被产生和分泌 主要通过调节性T细胞（Tcells）。虽然IL 35参与了许多细胞因子的调节， 细胞免疫应答，关于细胞因子是否真的存在，仍然存在重大的争论。这 争论是基于这样的事实，即试图从血液、生理溶液如腹水或 淋巴或甚至细胞培养上清液都失败了。我们最近免疫沉淀了Ebi 3和p35 用小鼠淋巴细胞的四跨膜蛋白CD 81抗体四跨膜蛋白是跨膜的 与细胞外小泡（外泌体）形成相关的蛋白质。这个有趣 这一观察促使我们检查IL 35是否以Ebi 3和p35蛋白的形式存在， 四跨膜蛋白作为外泌体分泌和获得。为了验证IL 35作为外泌体存在的想法， 具体目的1：为了测试外泌体是否能够抑制细胞因子的表达，我们设计了以下具体目的： 从tetraspanin敲除小鼠（CD 81，CD 63，CD 9）的耐受组的血清中分离的Ebi 3和p35缺失 阳性外泌体特异性目的2：检测来自耐受性-CD 81、CD 63或CD 9的淋巴细胞 敲除小鼠产生并释放功能性IL 35+免疫抑制性外来体。成功 完成本提案中概述的具体目标将填补我们对 细胞因子IL 35，并将理想地导致未来的应用，进一步我们的治疗理解， IL 35对人类的影响

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.