Longitudinal proteomics analysis in the immediate microenvironment of islet allografts during progression of rejection

Abstract

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.

Keywords: Allogeneic pancreatic islets; Allograft rejection; Allorejection; Anterior chamber of the eye; Immune biomarkers; Intraocular islet transplantation; Islet microenvironment; Localized proteomics; Type 1 diabetes (T1D).