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 December 1, 2011
Citation Information: J Clin Invest. 2012;122(1):119-131. https://doi.org/10.1172/JCI59285.
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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 2

Diabetogenic CTL arrest in postcapillary pancreatic venules.

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Diabetogenic CTL arrest in postcapillary pancreatic venules. (A and B) P...
(A and B) Pancreas sections from prediabetic RIP-LCMV.GP animals (n = 6) stained for CD8 and CD4. Autofluorescence shows general morphology. Red arrows indicate vascular structures with maximum diameter (μm). (C and D; corresponds to Supplemental Video 1) Vascular staining in naive animals. w/h = 1.52/d = 5/z = 32. (E) MIP image corresponds to Supplemental Video 2. w/h = 0.56/d = 4/z = 25. CTL can be seen in virtual arrest within postcapillary venules (red arrows). Inset represents a single Z-plane. (F) Cellular tracking shows immotile cells in the vascular bed (red arrows), lacking the “dragontails” that signify motility. (G) MIP image corresponds to Supplemental Video 3. w/h = 1.52/d = 5/z = 21. Setup analogous to E in another prediabetic animal. Green arrows indicate arrested cells in contact with the vascular wall. Cyan arrows show cells “crawling” against the vascular wall. White arrow shows endogenous “ghost” cells in arrest against the endothelium. Yellow arrow shows freely flowing transferred cell. Red arrow shows leakage of the vascular dye. (H) MIP image corresponds to Supplemental Video 4. w/h = 1.52/d = 5/z = 26. Setup analogous to E and G in another prediabetic animal. Green arrows point toward arrested transferred cells. (I) Image of the same region shown in H 8 minutes after dye injection demonstrates vascular leakage of the dextran–Texas Red dye (red arrows). Scale bars: 100 μm (A–E, G–I); 50 μm (F, insets in E, G).