PNNL

Abstract

While pancreatic islet transplantation can improve significantly the quality-of-life of patients with brittle type 1 diabetes, its applicability and benefits are limited due to various issues including the need to avoid immune-mediated rejection. In the present study, we used our experimental platform of allogeneic islet transplant in the anterior chamber of the eye (ACE-platform) to longitudinally monitor the rejection progression in mice and obtain aqueous humor samples representative of the immediate microenvironment of the graft for accurately-timed proteomic analyses before, during, and after the onset of rejection. LC-MS/MS based proteomics performed on such mass-limited small sample volumes (~5µL) identified a total of 1,296 proteins, and various clustering/enrichment analyses and predicted functional networks 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, whereas network prediction analyses revealed transition from humoral to cellular immune response and exacerbation of pro-inflammatory signaling during the progression of rejection. One of the proteins identified by our analyses of this aqueous humor-derived proteomics data as a candidate early biomarker of islet rejection was Cystatin 3, and it was validated by ELISA in parallel systemic (plasma) samples obtained from the rejecting mice. This study provides (1) experimental evidence demonstrating the feasibility of longitudinal local proteomics using small aqueous humor samples and (2) proof-of-concept for the discovery of early candidate biomarkers of impending immune attack from samples representing the immediate local microenvironment of ACE-transplanted islets that can be then further validated in systemic samples.