PRESENTED BY: Mollie Huber
|A goal of type 1 diabetes (T1D) research is to understand the complex islet microenvironment to determine the mechanisms of autoimmune-mediated beta cell dysfunction and loss. Here, we investigate the use of live pancreas tissue slices as a genuine and native three-dimensional microenvironment in the study of the islet-immune cell interface within the context of type 1 diabetes. In this system, endogenous tissue-resident or pathological immune cells can be studied in slices to identify translatable differences between donors at varying levels of T1D risk and development. Additionally, islet functionality at various disease stages can be assessed through Ca2+ imaging and dynamic insulin secretion assays. This approach allows for observations of islet health and function under insulitic conditions and can provide insights into disease mechanisms and progression.|
|Live human pancreas tissue slices were prepared from donors without diabetes or diabetes-relevant autoantibodies (ND, n=9), autoantibody-positive donors without a diagnosis of T1D (AAb+, n=5), and donors with short duration of 4 years or fewer T1D (T1D+, n=3) through methods co-developed by the nPOD/OPPC at the University of Florida and the Paul Langerhans Institute Dresden. Slices were imaged by time-lapse confocal microscopy on a Leica SP8 confocal equipped with an automated stage and incubation system. Live human pancreas tissue slices were stained with anti-CD3-APC and HLA-multimers to identify and track endogenous T cells within the tissue enabling the determination of in situ T cell activity around and within islets. Reflected light was used to locate islets due to their granularity from insulin content. An anti-ectonucleoside triphosphate diphosphohydrolase 3 (ENTPD3) antibody was used to identify islets with beta cells. Beta cell functionality was assessed through changes in intracellular Ca2+ concentrations through the use of Fluo-4 Ca2+ indicator dye. Overall, the viability and state of islets within the slices were determined through SYTOX Blue staining for viability, anti-ENTPD3 staining for beta cell identification, Ca2+ imaging for functionality, and staining with anti-CD3 and HLA multimers for T cell identification.|
In addition to human pancreas tissue slices, live mouse pancreas tissue slices were prepared from NOD-Rag1-/- and NOD-Rag1-/–AI4α/β TCR transgenic (NOD.AI4) mice. Endogenous T cells were stained with anti-CD3-APC and anti-CD8-APC and tracked in the tissue. Oregon Green 488 BAPTA-1 Ca2+ indicator dye was used to stain the mouse slices in order to record changes in intracellular Ca2+ concentrations. Perifusion experiments were also performed on the slices and insulin secretion traces will be obtained from hormone measurements using ELISAs.
Summary of Results
|Slices generated from ND donors had functional beta cells and few T cells in the immediate vicinity of most islets. An nPOD donor representative of this trend is case 6516 (ND). Islets from case 6516 exhibit strong Ca2+ responses following either 16.7 mM glucose or 30 mM KCl stimulations. Following perifusion, slices in this case produced insulin peaks following high glucose and KCl stimulations. Similarly, in AAb+ tissues, islets had strong Ca2+ responses to 16.7 mM glucose and KCl, as was noted in a GAD AAb+ case 6530. Again, insulin peaks during perifusion were recorded following high glucose and KCl stimulations. In contrast, islet functionality decreased in T1D+ slice cases. For example, T1D+ case 6533 had some T cell infiltrated islets with ENTPD3+ cells that were unresponsive to high glucose but maintained KCl responses. Within this same case, an ENTPD3+ islet without infiltrating T cells was observed that retained both high glucose and KCl responses. Insulin traces following perifusion from this case were abnormal with a small peak in low glucose and a strong peak following KCl stimulation, indicating dysfunctional glucose responsiveness, but residual insulin secretory capacity. This suggests a possible role for infiltrating T cells in beta cell dysfunction prior to beta cell killing.|
Additionally, T cell populations were identified more specifically through co-staining with antibodies and HLA multimers. Slice studies using these multimers identified IGRP and GAD reactive T cells in or near islets from slices of recent onset human T1D organ donors. When endogenous CD3+ cells were examined in live pancreas slices from a GAD AAb+ donor with T1D case 6469, one islet was found with >18 CD3+ T cells forming an apparent focal insulitis. To our knowledge, this is the first image of live, endogenous human immune cells attacking live insulin-producing beta cells from an autoantibody-positive organ donor with T1D in situ. Single cell RNAseq was applied to slices from T1D+ case 6472 and autoreactive T cell receptors specific to GAD were identified. Confirmatory studies using mouse pancreas tissue studies support the finding from human slices that the presence of T cells in the islet coincides with poor islet glucose sensitivity. Islets in slices from NOD.AI4 experiencing heavy insulitis exhibited no Ca2+ responses to high glucose. These same islets also were found to have lost all reflectivity. Conversely, islets from T1D-prone NOD.AI4 mice with few to no T cells as well as from non-diabetic NOD-Rag1-/- mice continued to have Ca2+ responses to both high glucose and KCl.
|Endogenous immune cell activity can be observed within live human and mouse pancreas tissue slices. Features such as in situ islets and endogenous immune cells remain intact. T cell behaviors and interactions in and around the islet can be recorded. The impacts of T cell activity on beta cells can be determined through functional studies such as Ca2+ recordings and perifusion experiments. Through the application of the slice method and the techniques discussed above, we have observed impaired beta cell function in response to glucose stimulation in the presence of T cells. Though this observation will need to be supported by future experiments, these studies will help to give a clear idea of what is occurring within the pancreas at different disease stages and the functional impacts on beta cells that these events cause. These studies will be critical in understanding if T cells cause beta cell dysfunction during T1D progression or only beta cell loss. This technology has strong potential as a fully human platform for understanding the etiopathology of T1D and testing interventional therapies that act in the local islet immune microenvironment. These initial successes are guiding our development of pancreas slices for further detailed three-dimensional studies of islet—immune cell interactions relevant to human T1D.|