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. 2011 Jan 25;108(4):1561-6.
doi: 10.1073/pnas.1018973108. Epub 2011 Jan 10.

Cellular and Molecular Events in the Localization of Diabetogenic T Cells to Islets of Langerhans

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Free PMC article

Cellular and Molecular Events in the Localization of Diabetogenic T Cells to Islets of Langerhans

Boris Calderon et al. Proc Natl Acad Sci U S A. .
Free PMC article

Abstract

Understanding the entry of autoreactive T cells to their target organ is important in autoimmunity because this entry initiates the inflammatory process. Here, the events that lead to specific localization of diabetogenic CD4 T cells into islets of Langerhans resulting in diabetes were examined. This was evaluated in two models, one in which T cells specific for a hen-egg white lysozyme (HEL) peptide were injected into mice expressing HEL on β cells and the other using T cells in the nonobese diabetic mouse strain, which develops spontaneous diabetes. Only T cells specific for β-cell antigens localized in islets within the first hours after their injection and were found adherent to intraislet dendritic cells (DCs). DCs surrounded blood vessels with dendrites reaching into the vessels. Localization of antigen-specific T cells did not require chemokine receptor signaling but involved class II histocompatibility and intercellular adhesion molecule 1 molecules. We found no evidence for nonspecific localization of CD4 T cells into normal noninflamed islets. Thus, the anatomy of the islet of Langerhans permits the specific localization of diabetogenic T cells at a time when there is no inflammation in the islets.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Localization of CD4 T cells to islets containing their antigen and MHC class II requirement. (A) Pooled results showing localization of activated 3A9 T cells into sublethally irradiated B10.BR and IP-HEL recipients at different times. Numbers of experiments are indicated on each bar. (B) Transfer of activated or unstimulated BDC T cells into NOD Rag1−/− recipients, analyzed at different time points. Numbers of experiments are indicated on each bar. (C) Localization of CD4 T cells into MHC class II-sufficient or -deficient mice. Activated BDC T cells were transferred into sublethally irradiated 8-wk-old NOD male mice (MHC II+/+) or MHC class II−/− deficient mice. The graph shows the number of infiltrated CD4 T cells per islet at 48 h after cell transfer. (D) Percentage of infiltrated islets from previous experiments (C). Data are represented as the mean (±SEM).
Fig. 2.
Fig. 2.
Localization of CD4 T cells to islets during partial inhibition by anti-class II MHC mAb. (A) T-cell distribution in islets 24 h after transfer of activated 3A9 T cells into IP-HEL mice treated as indicated, 1 d before cell transfer. The graph shows a representative experiment from a total of six, and each dot represents an islet. (B) Percentage of T cell-infiltrated islets at 24 and 48 h in IP-HEL mice treated as in A. Results are pooled from six and four experiments at 24 and 48 h, respectively. (C) T-cell distribution in islets 48 h after transfer of activated BDC T cells into NOD Rag1−/− mice treated as indicated. The graph shows a representative experiment from four experiments. (D) Percentage of T cell-infiltrated islets at 48 and 72 h in mice treated as in C. Results are pooled from four experiments. Data are represented as the mean (±SEM). P ≥ 0.05 [not significant (ns)]; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.
Fig. 3.
Fig. 3.
Partial inhibition of T-cell localization by anti-class II MHC and ICAM-1 mAb. (A) T-cell distribution in islets 24 h after transfer of activated 3A9 T cells into IP-HEL mice treated with a single mAb or a combination of both, 1 d before cell transfer. The graph shows a representative experiment from a total of two experiments. (B) Percentage of infiltrated islets from the previous experiment (A), showing the compiled data from two experiments. (C) T-cell distribution in islets 48 h after the transfer of activated BDC T cells into sublethally irradiated NOD.ICAM-1+/+ and NOD.ICAM-1−/− mice treated with a single anti-class II blocking mAb as indicated, 1 d before cell transfer. The graph shows a representative experiment from a total of two experiments. (D) Percentage of infiltrated islets from the previous experiment (C), showing the compiled data from two experiments at 48 h. Data are represented as the mean (±SEM). P ≥ 0.05 [not significant (ns)]; *P < 0.05; ***P < 0.001; ****P < 0.0001.
Fig. 4.
Fig. 4.
T cell-DC interaction in islets by two-photon microscopy and immunofluorescence. (A) Live isolated islet analysis evaluating percentage of 3A9 T cells found in contact with islet DCs at 24 h after cell transfer. T cell-DC contacts were obtained from a total of 20 examined islets from two independent experiments. 3A9 T cells were detected with anti-CD4 mAb, and DCs were detected with anti-CD11c mAb. (B) Plot shows T cell-DC contact durations of 3A9 (CFSE-labeled) T cells at 24 h after cell transfer in IP-HEL mice. Contact durations were obtained from 10- to 15-min time-lapse movies from two experiments. (C) Analysis showing percentage of T cell-DC remaining contact for any given duration. (D) Infiltrated islet at 24 h after activated T-cell transfer showing 3A9 T cells (red) and CD11c cells (green). (Inset) Micrograph showing contact of T cells with CD11c cells (yellow merged color). (Scale bar, 50 μm.)
Fig. 5.
Fig. 5.
Analysis of islet DCs in direct contact with blood vessels. (A) Two-photon microscopic analysis of a representative islet of C57BL/6 mice expressing YFP under the CD11c promoter (CD11c-YFP, shown in green) and endothelia stained with PECAM-1 (red). Reconstruction from a single stack (1-μm thickness). The arrow shows a DC in apposition to the vessel and dendrite, reaching into the lumen (yellow merging color). (B) 3D reconstruction by confocal microscopy of an islet from CD11c-YFP mice with vessels stained in red (PECAM-1) and CD11c-YFP (green). (Lower Left) Projection along the z axis (top view) from a stack of 30 optical sections (0.5-μm increments). (Upper Right) z–x and z–y reconstructions (side view) of same image stack (indicated as white lines). The image shows a DC dendrite inside the vessel (yellow merged color indicated by arrows). (C) Percentages of DCs contacting the vessel wall (not embedded), dendrites in the vessel wall, or DCs protruding into the lumen (n = 108). Data were obtained from 17 different visualized fields from two experiments. (D) Quantitative analysis of anti-class II mAb fluorescent bead (anti–I-Ak or anti–I-Ag7) localization. Each dot represents a single islet. The left and right y axes show the number of anti-class II beads per islet in B10.BR and NOD mice, respectively, representing compiled data from three independent experiments. **P < 0.01; ****P < 0.0001. (E) Localization of anti-class II–coated beads by immunofluorescence in islets of B10.BR mice previously injected i.v. with beads coated with anti–I-Ak. Islet DCs are shown in green (CD11c), blood vessels are shown in blue (PECAM-1), and fluorescent beads are shown in red. (Insets) Micrograph showing a representative islet DC in contact with a bead inside the lumen of the vessel. Bead and PECAM-1 (Bottom Right); CD11c and PECAM-1 (Middle Right); bead, CD11c, and PECAM-1 (Top Right). (Scale bar, 20 μm.)

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