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.

Figure 1. Establishment and characterization of a virus-induced diabetes model for in vivo pancreatic CTL imaging.

(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.

Figure 2. Diabetogenic CTL arrest in postcapillary pancreatic venules.

(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).

Figure 3. Profound leakiness of postcapillary islet venules is unrelated to endothelial apoptosis.

(A and B) Isosurfacing rendition of vascular staining from region depicted in Figure 2, H and I, before and 10 minutes after injection of the dye, respectively. w/h = 1.52/d = 5/z = 26. (C and D) Naive control animal imaged before and 14 minutes after injection with dextran–Texas Red (dextran-TxRed). w/h = 1.52/d = 5/z = 33. (E) Quantitative analysis of vascular leakiness as observed in A and B compared with baseline in C and D. Leakiness was defined as increase in isosurfaced dextran–Texas Red–derived fluorescent signal and was normalized to T = 0 immediately after injection. Representative of observations from 3 individual prediabetic animals and 3 controls. (F and G) TUNEL staining was performed to reveal ongoing apoptosis in the transfer model during the late prediabetic phase. TUNEL⁺ nuclei are exclusively observed within the β cell mass and inflammatory infiltrate. Red arrows indicate vascular structures and the lack of ongoing apoptosis within these regions. Images are representative of at least 3 consecutive sections from 4 individual prediabetic animals. Scale bars: 100 μm.

Figure 4. CTLs exhibit random walk motility in the exocrine pancreas.

(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).

Figure 5. Unimpeded but nondirectional CTL migration between anatomically nearby islets.

(A and B) During imaging, CTLs were often found to establish a 3D continuum between adjacent islets. A and B represent 3 adjacent islets (numbered islet 1, 2, and 3) captured in 2 individual Z-stacks over time. B corresponds to Supplemental Video 9. (C–E) 3D regions of interest were drawn in these inter-islet zones, and cellular motility was analyzed within these regions. D corresponds to Supplemental Video 10. Red lines show linear regression plot. Image dimensions: A, w/h = 1.52/d = 6/z = 22; B, w/h = 0.70/d = 6/z = 22; C, w/h = 1/d = 6/z = 22; D, w/h = 1.11/d = 6/z = 16; E, w/h = 1.51/d = 5/z = 28. Horizontal bars are means and associated error bars represent the SEM. Scale bars: 100 μm.

Figure 6. CTL motility within pancreatic islets imaged at cellular resolution.

(A–C) Pancreatic islet (green, GFP) and infiltrating CTL (magenta, CFP) populations from 3 individual mice were imaged at the highest digital magnification that would fully encompass β cell mass and associated CTLs. Dimensions: A, w/h = 0.79/d = 5/z = 22; B, w/h = 0.88/d = 5/z = 20; C, w/h = 0.91/d = 5/z = 25. Acquisition times are 10 minutes, 29 minutes, and 14 minutes, respectively, with t = 60 s. A corresponds to Supplemental Video 11. Five representative, high-resolution examples are included for each series that document CTL–β cell interactions (first frame [T1 in min] and last frame of contact [T2 in min]). Supplemental Videos 12 and 13 correspond to magnified zones from A and B, respectively. (D) Enlarged region from C showing a β cell that is almost entirely engulfed by CTLs. (E) Control experiment using mice with fluorescently labeled but antigen-deficient β cells. A small exocrine population of CTLs can be observed. One frame is shown from a 14-minute sequence during which none of the CTLs shown here infiltrated the islet (representative of duplicate control experiments). Dimensions: w/h = 0.75/d = 5/z = 24. (F–I) Kinetic CTL population parameters obtained via 3D cellular-tracking analysis in Imaris. Color coding correlates with the image sequences in A–C. Curves in I were generated via nonlinear regression analysis in GraphPad and, in combination with R² values, reveal deviation from linearity and thus constrained motility of the intra-islet CTL population. Horizontal bars are means and associated error bars represent the SEM. Scale bars: 80 μm (A–C and E); 10 μm (D).

Figure 7. β cell death and CTL motility after antigen depletion.

(A–C) Events of β cell death with loss of GFP as a readout measure (red arrows). A corresponds to Supplemental Video 14. B is derived from the same sequence used in Figure 5C. Imaging volume V and number of unique CTL tracks are indicated. Dimensions: A, w/h = 0.58/d = 6/z = 21; B, w/h = 1/d = 6/z = 22; C, w/h = 1.08/d = 6/z = 10; t = 30 s for all sequences. (D) Representative control islet from naive animal imaged at maximum laser intensity; w/h = 0.51/d = 5/z = 22. (E) Association of arrested CTL (CFP, pseudocolored magenta; white arrows) with dying β cells (GFP, green; red arrows). E corresponds to Supplemental Videos 15 and 16; w/h = 0.86/d = 5/z = 22. (F–I) Representative images from full-blown diabetic mice and corresponding tracking results. Imaging volume V and number of unique CTL tracks are indicated. Supplemental Video 17 shows an enlarged region of F, while G corresponds to Supplemental Video 18, which includes vascular staining. Representative of 3 individual experiments. Dimensions: F, w/h = 1.52/d = 5/z = 20; G, w/h = 1.52/d = 5/z = 18. Yellow arrows in F and G indicate remaining β cells. P values were obtained with Mann-Whitney analysis. Horizontal bars are means and associated error bars represent the SEM. Scale bars: 100 μm (B, C, E–G); 50 μm (A and D).