The availability of pancreas slices from nPOD from T1D and normal control donors has provided a huge thrust in the study of T1D islet biology as it requires the inflamed islet to be intact within a pancreas slice for accurate assessment. We have developed capabilities to culture these pancreas slices for ~10 days allowing virus gene transfer of organelle-specific fluorophores and perform live-cell imaging to track islet cell secretory function (calcium, exocytosis) and potentially other organelle disruption (mitochondria, autophagy). We plan to use these capabilities on the following 3 aims.
Aim 1: Assess β-β cell and α-α cell connectivity that we hypothesize to be differentially perturbed between early onset Aab+ islets vs late-stage T1D, which can disrupt their secretory function; and test potential agents that can rectify these connectivity defects and improve regulation of secretion. β-cells and α-cells will have cell-targeted expression of a calcium and exocytosis fluorophores as reporters for secretory function.
Aim 2: Assess how engineered human T cells (Tregs, CD4 and CD8) that interact with islets (β-cell and α-cell) to cause initial disruption of function progressing to β-cell killing and α−cell dysfunction (causing glucose blindness). Tcells will be tagged with red fluorophore and β- (or α-) cell will be tagged with green fluorophores to visualize their interactions in real time and at high spatial resolution. This is a collaboration with KQ5 group – Qizhi Tang.
Aim 3. Assess how pancreatic duct defects or deficiency of CFTR cause islet dysfunction. We will knockdown CFTR expression with an adenovirus in human pancreas slices and assess the islet cell function by functional imaging as described above. This is in collaboration with KQ3: role of exocrine pancreas in T1D; and is in collaboration with Rebecca Hull (Seattle and Edmonton).