The applicability and benefits of pancreatic islet transplantation are limited due to various issues including the need to avoid immune-mediated rejection. Here, we used our experimental platform of allogeneic islet transplant in the anterior chamber of the eye (ACE-platform) to longitudinally monitor the progress of rejection in mice and obtain aqueous humor samples representative of the microenvironment of the graft for accurately-timed proteomic analyses. LC-MS/MS-based proteomics performed on such mass-limited samples (~5 μL) identified a total of 1296 proteins. Various analyses revealed distinct protein patterns associated with the mounting of the inflammatory and immune responses and their evolution with the progression of the rejection. Pathway analyses indicated predominant changes in cytotoxic functions, cell movement, and innate and adaptive immune responses. Network prediction analyses revealed transition from humoral to cellular immune response and exacerbation of pro-inflammatory signaling. One of the proteins identified by this localized proteomics as a candidate biomarker of islet rejection, Cystatin 3, was further validated by ELISA in the aqueous humor. This study provides (1) experimental evidence demonstrating the feasibility of longitudinal localized proteomics using small aqueous humor samples and (2) proof-of-concept for the discovery of biomarkers of impending immune attack from the immediate local microenvironment of ACE-transplanted islets. SIGNIFICANCE: The combination of the ACE-platform and longitudinal localized proteomics offers a powerful approach to biomarker discovery during the various stages of immune reactions mounted against transplanted tissues including pancreatic islets. It also supports proteomics-assisted drug discovery and development efforts aimed at preventing rejection through efficacy assessment of new agents by noninvasive and longitudinal graft monitoring.

中文翻译：

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在排斥反应进展过程中，胰岛同种异体移植物的直接微环境中的纵向蛋白质组学分析。

由于包括避免免疫介导排斥反应在内的各种问题，胰岛移植的适用性和益处受到限制。在这里，我们使用同种异体胰岛移植在眼前房中的实验平台（ACE平台）纵向监测小鼠排斥反应的进程，并获得代表移植物微环境的房水样品以进行准确的蛋白质组分析。在此类质量受限样品（〜5μL）上进行的基于LC-MS / MS的蛋白质组学鉴定出总共1296种蛋白质。各种分析揭示了与炎症和免疫反应的增强相关的独特蛋白质模式，以及它们随着排斥反应的进展而发展的过程。途径分析表明，细胞毒性功能，细胞运动，以及先天性和适应性免疫反应。网络预测分析揭示了从体液免疫反应到细胞免疫反应的转变以及促炎性信号的加剧。通过该蛋白质组学鉴定为胰岛排斥的候选生物标志物的蛋白之一，胱抑素3，通过房水中的ELISA进一步验证。这项研究提供（1）实验证据，证明使用小型房水样本进行纵向局部蛋白质组学的可行性，以及（2）从ACE植入的胰岛的局部局部微环境中发现即将发生的免疫攻击的生物标记的概念验证。意义：ACE平台和纵向局部蛋白质组学的结合为在针对包括胰岛在内的移植组织的免疫反应的各个阶段中发现生物标志物提供了一种有力的方法。它还支持蛋白质组学辅助药物的发现和开发，旨在通过无创和纵向移植物监测对新药进行疗效评估，从而防止排斥反应。