Intravital imaging of CTLs killing islet cells in diabetic mice
Ken Coppieters, Natalie Amirian, Matthias von Herrath
Ken Coppieters, Natalie Amirian, Matthias von Herrath
Published January 3, 2012
Citation Information: J Clin Invest. 2012;122(1):119-131. doi:10.1172/JCI59285.
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Categories: Research Article Autoimmunity

Intravital imaging of CTLs killing islet cells in diabetic mice

Abstract

Type 1 diabetes (T1D) is caused by autoimmune destruction of the insulin-producing β cells in the pancreatic islets, which are essentially mini-organs embedded in exocrine tissue. CTLs are considered to have a predominant role in the autoimmune destruction underlying T1D. Visualization of CTL-mediated killing of β cells would provide new insight into the pathogenesis of T1D, but has been technically challenging to achieve. Here, we report our use of intravital 2-photon imaging in mice to visualize the dynamic behavior of a virally expanded, diabetogenic CTL population in the pancreas at cellular resolution. Following vascular arrest and extravasation, CTLs adopted a random motility pattern throughout the compact exocrine tissue and displayed unimpeded yet nonlinear migration between anatomically nearby islets. Upon antigen encounter within islets, a confined motility pattern was acquired that allowed the CTLs to scan the target cell surface. A minority of infiltrating CTLs subsequently arrested at the β cell junction, while duration of stable CTL–target cell contact was on the order of hours. Slow-rate killing occurred in the sustained local presence of substantial numbers of effector cells. Collectively, these data portray the kinetics of CTL homing to and between antigenic target sites as a stochastic process at the sub-organ level and argue against a dominant influence of chemotactic gradients.

Authors

Ken Coppieters, Natalie Amirian, Matthias von Herrath

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Figure 1

Establishment and characterization of a virus-induced diabetes model for in vivo pancreatic CTL imaging.

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Establishment and characterization of a virus-induced diabetes model for...
(A) Recipient mice expressing GFP and viral antigen on their β cells were injected with purified, naive, fluorescently labeled P14 CD8+ T cells 1 day prior to infection with LCMV. (B) Supplementation with TCR transgenic effectors results in a consistent synchronization of hyperglycemia onset as compared with the conventional model. (C) 2-Photon imaging was successful in capturing β cell mass and infiltrating CTL ideally on days 7 and 8 after infection, immediately prior to diabetes onset. Vascular staining (here pseudocolored in red) reveals the local (micro-)vascular circuit. w/h = 1.52/d = 5/z = 41. Scale bar: 100 μm. (D) Detailed immunofluorescent analysis of the cellular composition of the islet infiltrate at various stages of the diabetogenic process. Upper panel demonstrates validity of the stainings on frozen spleen sections. Transferred CD8+ T cells were detected by way of staining for the congenic marker CD45.1. Images represent consecutive sections of islets obtained at days 0, 8, and 10 after infection and are representative of 4 animals analyzed at each time point. Red outline defines approximate islet contours based on DAPI morphology for ease of interpretation. Scale bar: 100 μm.