Beta cells transfer vesicles containing insulin to phagocytes for presentation to T cells

Abstract

Beta cells from nondiabetic mice transfer secretory vesicles to phagocytic cells. The passage was shown in culture studies where the transfer was probed with CD4 T cells reactive to insulin peptides. Two sets of vesicles were transferred, one containing insulin and another containing catabolites of insulin. The passage required live beta cells in a close cell contact interaction with the phagocytes. It was increased by high glucose concentration and required mobilization of intracellular Ca2+. Live images of beta cell-phagocyte interactions documented the intimacy of the membrane contact and the passage of the granules. The passage was found in beta cells isolated from islets of young nonobese diabetic (NOD) mice and nondiabetic mice as well as from nondiabetic humans. Ultrastructural analysis showed intraislet phagocytes containing vesicles having the distinct morphology of dense-core granules. These findings document a process whereby the contents of secretory granules become available to the immune system.

Keywords: autoimmune diabetes; autoimmunity; insulin reactivity; insulin-reactive T cells.

Fig. 1.

Beta cells transfer immunogenic insulin to phagocytes. Flow cytometry plots of isolated endocrine cells (A) or intraislet macrophages (B) stained with antibodies reactive to insulin and to the B:9–23 peptide. Cells were gated as indicated in the panels. Red histograms correspond to the isotype control antibody staining. Blue histograms are either antiinsulin or anti-B:9–23 peptide staining. Numbers in each plot indicate the mean fluorescence intensity for their respective histogram. (C) Secretory granules were isolated by differential centrifugation from beta cells isolated from NOD.Rag1^−/− mice and offered to spleen DCs, and the response of the 8F10 T cell or IIT-3 T cell was then assayed. Shown are the responses to the 5,000 and 25,000 × g fractions (in 5K and 25K, respectively) and as a control to the B:9–23 peptide. (D) The characterization of the two CD4 T cells to insulin (8). The FLT-3L DCs were incubated with insulin or with the B:9–23 peptide, each at 10 μM. 8F10 reacts with peptides B:9–23 or B:12–20 (sequence shown below the graph) but not with insulin or B:13–21. IIT-3 reacts with insulin and peptides B:9–23 and B:13–21. (E) A representative assay (of n > 25). Indicated are the cells used in the assay. Beta cells were from 6-wk-old NOD.Rag1^−/− mice; the APCs were DCs obtained from the spleen of mice previously injected with FLT-3L. Background response of the T cells never exceeds 150 cpm. (F) Summary of the first series of experiments. The explanation is in the text. (G) Antibody to I-A^g7 inhibits the transfer. The culture included the presence or absence of 10 μg/mL of the antibody Ag2.42.67 specific for I-A^g7. (H) Same as in A but testing islets from B6 mice. Shown is a representative experiment of two experiments. (I) As in A but testing human islets. The results are pooled from two experiments.

Fig. 2.

Transfer requirements and life of the peptide–MHC complex. (A) Same as in Fig. 1E but adding one variable: the separation of beta cells and DCs by a 0.4-μm polycarbonate filter, which results in the lack of transfer. (B) Examination of dead beta cells after streptozotocin (STZ) treatment, 50 mM overnight, showing a complete lack of transfer. (C) DCs were treated with chloroquine (100 μM) for 2 h and washed extensively to remove the drug, and then the DCs were incubated with the beta cells for 4 h and tested with the T cells. (D) Control experiments using DCs incubated with insulin or peptide in the presence of chloroquine. (E) DCs were incubated with beta cells for 1 h, after which the DCs were separated. T-cell hybridomas were added to the separated DCs at the indicated times; the 100% numbers for 8F10 and IIT-3 are 5,030 cpm and 20,447 cpm, respectively.

Fig. 3.

Effects of thapsigargin and evaluation of Ca²⁺ requirements. (A) Beta cells were treated with thapsigargin (10 μM) for 12 h in media with 5 or 25 mM glucose, after which the drug was removed and DCs and T cells were added to the culture. The experiment is representative of 10 experiments. (B) The beta cells were cultured in the absence of extracellular Ca²⁺, either in media prepared without Ca²⁺ or media containing 10 μM EGTA–. The results were identical and have been pooled. The T cells were IIT-3; mean cpm at 5 mM glucose was 5,686; at 25 mM, glucose was 11,738. (C and D) Both beta cells and DCs were treated with BAPTA-AM for 1 h, and then the cells were separated. The passage to the DCs was then probed with IIT3. In C and D, cells were in media with 5 mM or 25 mM glucose, respectively. Results are representative of two experiments. In the lower portion of the graph, the DCs were treated with BAPTA for the same time, after which the drug was washed off and insulin was added. The processing of insulin was consistently inhibited by about 30%.

Fig. S1.

ERS markers evaluation. Beta cells from NOD.Rag1^−/− mice were incubated in high or low glucose media for 1 h, 4 h, and 24 h. cDNA was synthesized from extracted mRNA. Hspa5 (BIP), Wfs1, Ddit3 (CHOP), Ppp1r15a (GADD34), and Atf4 were amplified with specific primers by quantitative RT-PCR. The fold change in gene expression was calculated using 2^–ΔCT. Bars are mean ± SD of biological duplicates. Thapsigargin (Tg), 0.1 μM, was a positive control for ERS induction. Results are representative of three experiments.

Fig. 4.

Imaging and electron microscopy. (A) DCs labeled with CellTrace Violet (red) were added to NIT GFP-ZnT8-Insulinomas. Left panel (Movie S1) shows the contact area of NIT insulinomas in green with the DCs. Note the accumulation of GFP⁺ granules at the contact area. No movement of granules was observed in BAPTA/AM-treated NIT cells (Right panel and Movie S2). (B–E) Electron micrographs. B represents an islet from an 8-wk-old female NOD; C–E are islets taken from NOD.Rag1^−/− mice at 14 wk of age. The arrow in B indicates a vessel. In the enlarged area, one of the dense-core granules is indicated by an arrow. In panel D a phagocyte is shown in between beta cells, and an arrow points to a dense-core granule. Panel E shows a portion of a phagocyte with endocytosed material in the form of vesicles containing an electron-dense core, with others containing amorphous content.

Fig. S2.

(A and B) Granule accumulation in insulinomas at the contact side of DCs (“synapse”). A is a 3D reconstruction and computational sectioning of DCs at the beginning of synapse formation (Left) and at the point of a mature synapse (Right) (related to Fig. 4 and Movie S1). Green particles increase dramatically inside the DCs during the process of synapse generation (white arrows). B is a quantitation of synapse per DC at 30 min.