Antigen recognition in the islets changes with progression of autoimmune islet infiltration - PubMed (original) (raw)

. 2015 Jan 15;194(2):522-30.

doi: 10.4049/jimmunol.1400626. Epub 2014 Dec 10.

Kaitlin Corbin 2, Ashley Mahne 3, Bonnie E Levitt 4, Matthew J Gebert 4, Eric J Wigton 4, Brenda J Bradley 1, Kathryn Haskins 1, Jordan Jacobelli 1, Qizhi Tang 3, Matthew F Krummel 2, Rachel S Friedman 5

Affiliations

Antigen recognition in the islets changes with progression of autoimmune islet infiltration

Robin S Lindsay et al. J Immunol. 2015.

Abstract

In type 1 diabetes, the pancreatic islets are an important site for therapeutic intervention because immune infiltration of the islets is well established at diagnosis. Therefore, understanding the events that underlie the continued progression of the autoimmune response and islet destruction is critical. Islet infiltration and destruction is an asynchronous process, making it important to analyze the disease process on a single islet basis. To understand how T cell stimulation evolves through the process of islet infiltration, we analyzed the dynamics of T cell movement and interactions within individual islets of spontaneously autoimmune NOD mice. Using both intravital and explanted two-photon islet imaging, we defined a correlation between increased islet infiltration and increased T cell motility. Early T cell arrest was Ag dependent and due, at least in part, to Ag recognition through sustained interactions with CD11c(+) APCs. As islet infiltration progressed, T cell motility became Ag independent, with a loss of T cell arrest and sustained interactions with CD11c(+) APCs. These studies suggest that the autoimmune T cell response in the islets may be temporarily dampened during the course of islet infiltration and disease progression.

Copyright © 2015 by The American Association of Immunologists, Inc.

PubMed Disclaimer

Figures

Figure 1

Figure 1. Intra-vital imaging maintains intact blood flow without damaging the pancreas

A) Setup for intra-vital 2-photon pancreas imaging. A heated suction window stabilizes the surgically exposed pancreas for imaging. B–C) Representative maximum intensity projection images of islets imaged intra-vitally through the suction imaging window captured using 2-photon microscopy. Vascular space is labeled with 70kD dextran-rhodamine (red). Images are representative of 7 experiments. B) Islets are identifiable by their dense convoluted vasculature compared to exocrine tissue vasculature. The border of the islet is identified with a yellow dotted line. Scale bar = 30μm. C) NOD mouse islet with transferred BDC-2.5 T cells (green). The collagen fluorescence is provided by the second harmonic (blue) which demonstrates that the T cell infiltration is inside the islet basement membrane. Scale bar=100μm. D) Neutrophils do not accumulate at the site of imaging. Fluorescently labeled neutrophils were transferred into mice prior to surgical exposure and imaging of the pancreas through the suction window. The number of neutrophils was counted every ninety seconds. The lack of neutrophil accumulation shows that the imaging site was not damaged during imaging. Data are representative of 1 islet per mouse in 3 experiments. E) Suction imaging window does not impede blood flow. Fluorescent beads were tracked within blood vessels of different diameter within and around the pancreatic islets. Each dot represents one bead. Data are representative of 3 islet per mouse in 2 experiments.

Figure 2

Figure 2. Explanted islet imaging allows high resolution, high throughput imaging

A) Setup of explanted islet 2-photon imaging. Isolated pancreatic islets were mounted in low melting temperature agarose and maintained at 35–37°C with constant flow of oxygenated media. B) Representative multiple field image of explanted islets. BDC-2.5 T cells (red) were transferred into WT NOD recipients and islets were isolated. Image shows a maximum intensity projection of explanted islets. Nuclei are labeled with Hoechst 33342 (blue). Scale bar=200μm. Images are representative of >3 experiments.

Figure 3

Figure 3. T cell motility increases with progression of islet infiltration

A–B) Activated BDC-2.5 T cells (green) were fluorescently labeled and transferred 24h prior to imaging. Representative maximum intensity projection images from explanted (A) or intra-vital (B) islets captured using 2-photon microscopy. Dashed lines represent the islet border. Green lines represent 10 minute paths of BDC-2.5 T cell movement. Images are representative of islets with mild infiltration (less than 30% of islet volume infiltrated) or advanced infiltration (30–60% of islet volume infiltrated). Scale bars= 50μm. C–E) Quantification of T cell motility within explanted or intra-vital islets. Data pooled from 16 explanted islets from 4 mice in 3 independent experiments and 16 intra-vital islets from 7 mice in 7 independent experiments. *=P<0.05, **=P<0.01; measured by Student’s t test. C) Linear correlation of the average T cell velocity within an islet versus the percentage of the infiltrated islet volume. Each dot represents the average of all of the tracked T cells within a single islet. D–F) Average of individual islets. D) T cell crawling speed in explanted vs. intra-vital islets. E) Fold increase in crawling speed between islets with mild and advanced infiltration.

Figure 4

Figure 4. T cells reduce arrest and increase displacement as islet infiltration progresses

Activated BDC-2.5 T cells were fluorescently labeled and transferred 24 hours prior to imaging as in Figure 3. Data were pooled from 16 explanted islets from 4 mice in 3 independent experiments and 16 intra-vital islets from 7 mice in 7 independent experiments. *= P<0.05, **= P<0.01, ***= P<0.001; measured by 2-way Anova with Bonferroni posttests or Student’s t test. A–B) Average of individual islets. A) T cell track straightness (1= cell moves in a straight line). B–C) T cell arrest coefficient (% of time crawling speed is <2μm/min). D) Mean squared displacement (μm2) over time.

Figure 5

Figure 5. Early T cell arrest is antigen dependent

BDC-2.5 T cells were fluorescently labeled and transferred into WT NOD or NOD.C6 recipient mice 48 hours prior to imaging to determine infiltration state. BDC-6.9 T cells and BDC-2.5 T cells were co-transferred 24 hours prior to imaging to determine T cell motility. The antigen for BDC-6.9 T cells is absent in the NOD.C6 recipients. Data represent 25 wild type islets from 4 mice in 4 experiments and 25 NOD.C6 islets from 5 mice in 5 experiments. Each point represents the average T cell motility within 1 islet. **= P<0.01, ***= P<0.001 by two-tailed Student’s t test. A) In WT NOD islets where the antigen was present for BDC-2.5 and BDC-6.9 T cells, both types of T cells increase motility at a similar rate as islet infiltration increases. B) In NOD.C6 islets, where the antigen is present for BDC-2.5 T cells, but absent for BDC-6.9 T cells, the BDC-6.9 T cells move faster in the absence of their antigen. C) The ratio of average BDC-6.9 T cell motility to the average BDC-2.5 T cell motility within the same islet. Infiltration states: very mild (0–5%), mild (5–30%), and advanced (30–60%). D) Comparison of the arrest coefficient of all BDC-2.5 and BDC-6.9 T cells within islets with mild infiltration from NOD.C6 mice. BDC-6.9 T cells (No Ag) have reduced arrest.

Figure 6

Figure 6. Sustained T cell-CD11c+ APC interactions are lost with progression of islet infiltration

Fluorescently labeled BDC-2.5 T cells were transferred into CD11c-YFP hosts 24 hours prior to islet isolation and imaging. Data represent 15 islets from 5 mice in 5 independent experiments. ***= P<0.001 by two-tailed Student’s t test. A–B) Maximum intensity projection images showing BDC-2.5 (red) and CD11c+ APCs (green) within pancreatic islets. Yellow box indicates the region shown in time-lapse images on the right. Grey circles highlight the CD11c+ APCs that the T cell of interest has contacted; yellow arrows show current T cell-APC contacts. Time stamps= min:sec. A) Sustained T cell-APC interaction in an islet with mild infiltration. Scale bar= 40μm for whole islet and 10μm for time-lapse images. B) Transient T cell contacts with different CD11c+ APCs in an islet with advanced infiltration. Scale bar= 50μm for whole islet and 20μm for time-lapse images. C) Average percentage of T cells within individual islets that contact CD11c+ APCs for at least 2 min. D) Average percentage of T cells that contacted CD11c+ APCs, which had sustained interactions of ≥ 10 min. E) Duration of T cell- CD11c+ APC contacts.

Similar articles

Cited by

References

    1. Gagnerault MC, Luan JJ, Lotton C, Lepault F. Pancreatic Lymph Nodes Are Required for Priming of Beta Cell Reactive T Cells in NOD Mice. J Exp Med. 2002;196:369–377. - PMC - PubMed
    1. Bluestone JA, Herold K, Eisenbarth G. Genetics, pathogenesis and clinical interventions in type 1 diabetes. Nature. 2010;464:1293–1300. - PMC - PubMed
    1. Driver JP, Serreze DV, Chen YG. Mouse models for the study of autoimmune type 1 diabetes: a NOD to similarities and differences to human disease. Semin Immunopathol. 2011;33:67–87. - PubMed
    1. Jansen A, Homo-Delarche F, Hooijkaas H, Leenen PJ, Dardenne M, Drexhage H. Immunohistochemical characterization of monocytes-macrophages and dendritic cells involved in the initiation of the insulitis and beta-cell destruction in NOD mice. Diabetes. 1994;43:667–675. - PubMed
    1. Penaranda C, Tang Q, Ruddle N, Bluestone J. Prevention of diabetes by FTY720-mediated stabilization of peri-islet tertiary lymphoid organs. Diabetes. 2010;59(6):1461–1468. - PMC - PubMed

Publication types

MeSH terms

Substances

Grants and funding

LinkOut - more resources