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 4

CTLs exhibit random walk motility in the exocrine pancreas.

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CTLs exhibit random walk motility in the exocrine pancreas. (A–E) Images...
(A–E) Images from time-lapse sequences with tracked CTLs in magenta. Green clusters represent β cell mass. Tracks identify past 5 cell positions. Only cells that were tracked in 5 consecutive frames were included for analysis. C corresponds to Supplemental Video 6 and E with Supplemental Video 7. (F; corresponds to Supplemental Video 8) Analogous setup and analysis but using in vivo peptide/ adjuvant activation (ref. 31). Red lines show linear regression plot. (G) CTL stratification strategy for chemotaxis analysis according to distance from islet centroid. Red arrows represent net CTL displacement, green is isosurfaced islet contour from which centroid position was derived, and black lines represent stratification groups. (H) Net displacement relative to islet centroid, categorized according to initial position from centroid (μm). (I) Plot of track straightness across stratified CTL. (J) Comparison of track straightness between CTLs that display net inward versus outward movement relative to the islet centroid. H–J represent pooled data from 3 individual time sequences; no significant differences were found using 1-way ANOVA. Dimensions: A, w/h = 0.91/d = 6/z = 10; B, w/h = 1.52/d = 6/z = 24; C, w/h = 0.52/d = 6/z = 17; D, w/h = 0.80/d = 6/z = 15; E, w/h = 0.27/d = 5/z = 24; F, w/h = 0.69/d = 6/z = 14; t = 30 s for all series. Graphs show means ± SEM. Scale bars: 50 μm (C–G); 100 μm (A and B).