Background

Type 1 Diabetes (T1D) is an autoimmune disease affecting the insulin-producing beta cells of the pancreatic islet. Autoimmune attack is precipitated by a combination of genetic and environmental factors very early in childhood, and leads to a decline in beta cell function during a preclinical asymptomatic phase. The disease then manifests clinical symptoms upon the onset of beta cell death. Although T1D-associated autoimmunity is composed of both an earlier, non-specific innate and subsequent human leukocyte antigen-linked adaptive immune response, the innate immune response is of particular interest because its severity has been inversely correlated with the maturation state of pancreatic beta cells. However, it is unclear whether immaturity attenuates the innate immune response, or whether the response itself de-differentiates vulnerable cells. To determine the direction of causality, this project aims to optimize a cytokine cocktail to model innate immune attack on hPSC-generated pancreatic islet organoids, which can be entrained by a circadian feed-fast cycle to reach varying stages of maturity. 

Methods

Although the proposed model system for later experiments are hPSC-generated pancreatic islet organoids, optimization assays were performed in the Min6 mouse insulinoma beta cell line to minimize costs. Three candidate cytokine cocktails were identified based on interferon signaling pathways leading to apoptosis and other pro-inflammatory responses. To assess apoptosis and late-stage death from cytokine treatment, 3D Min6 cells were stained with Annexin-V and propidium iodide respectively, and quantified by fluorescence activated cell sorting (FACS). ER stress associated with pro-inflammatory responses and enhanced secretory burden was assayed by an immunofluorescence (IF) assay on 2D Min6 cells. 

Results 

Min6 cells showed little increase in cell death when treated with any cytokine cocktail. However, although FACS failed to quantify levels of ER stress markers, 2D Min6 cells showed an increased degree of ER stress when treated with a cytokine cocktail. 

Conclusions 

Min6 cells are responsive to cytokine treatment in terms of ER stress but not apoptosis. An optimal cytokine cocktail cannot yet be determined. Future assays should be performed in pancreatic islet organoids.