Amplification of autoimmune response through induction of dendritic cell maturation in inflamed tissues

Abstract

Dendritic cells (DCs) are essential in T cell-mediated destruction of insulin-producing beta cells in the islets of Langerhans in type 1 diabetes. In this study, we investigated T cell induction of intra-islet DC maturation during the progression of the disease in both autoimmune-prone NOD and resistant C57BL/6 mice. We demonstrated steady-state capture and retention of unprocessed beta cell-derived proteins by semimature intra-islet DCs in both mouse strains. T cell-mediated intra-islet inflammation induced an increase in CD40 and CD80 expression and processing of captured Ag by resident DCs without inducing the expression of the p40 subunit of IL-12/23. Some of the CD40(high) intra-islet DCs up-regulated CCR7, and a small number of CD40(high) DCs bearing unprocessed islet Ags were detected in the pancreatic lymph nodes in mice with acute intra-islet inflammation, demonstrating that T cell-mediated tissue inflammation augments migration of mature resident DCs to draining lymph nodes. Our results identify an amplification loop during the progression of autoimmune diabetes, in which initial T cell infiltration leads to rapid maturation of intra-islet DCs, their migration to lymph nodes, and expanded priming of more autoreactive T cells. Therapeutic interventions that intercept this process may be effective at halting the progression of type 1 diabetes.

FIGURE 1

Phenotype of NOD.MIP-GFP mice. B6.MIP-GFP transgenic mice were backcrossed to the NOD background. A, Diabetes incidence of NOD.MIP-GFP mice and their transgene-negative littermates at nine generations of backcrossing are shown. B, Immunohistological analysis of islets in NOD.MIP-GFP transgenic mice and in transgene-negative littermates at eight generations of backcrossing. The β cells are stained deep purple by aldehyde fuchsin staining. The β, δ, and polypeptide-producing cells are identified by the brown immunohistochemical staining for glucogon, somatostatin, and pancreatic polypeptide, respectively. C, Insulitis score of NOD.MIP-GFP transgenic mice and their transgene-negative littermates at 9–15 wk of age. Numbers of islets analyzed are shown in the category x-axis label.

FIGURE 2

Location and numbers of intraislet DCs. Islets were isolated from NOD.CD11c-YFP.Rag2^−/− and B6.CD11c-YFP mice, stained with CMTMR or Hoechst 33342, and analyzed by microscopy. A, Maximal z projection of a representative objective field of B6.CD11c-YFP islet imaged. B, Enumeration of numbers of DCs in islets. Each circle represents one islet, and the lines represent mean ± SD (NOD.CD11c-YFP.Rag2^−/−, n = 82, mean = 7.65, SD = 7.51; B6.CD11c-YFP, n = 72, mean = 7.75, SD = 7.18).

FIGURE 3

Phenotype of intra-islet DCs in steady state. Single-cell suspension of islets and PLNs isolated from NOD.Rag2^−/− or B6 mice were analyzed by flow cytometry to determine the cell surface markers expressed by intra-islet DCs. A, Characterization of subtypes of intra-islet DCs. Events shown are gated on live cells (4′,6-diamidino-2-phenylindole (DAPI)⁻). B, Expression of MHC class II and costimulatory molecules expressed on intra-islet DCs (gated on CD45⁺DAPI⁻CD11c⁺ cells, bold lines) in comparison with DCs in the PLN (shaded histograms). The result is representative of at least two independent experiments in each of the mouse strains.

FIGURE 4

Intra-islet DCs in MIP-GFP transgenic mice acquire and maintain intact GFP. Flow cytometric analysis of GFP expression by intra-islet and lung DCs in NOD.Rag2^−/−.MIP-GFP (A) and B6.MIP-GFP (B) mice. Events shown are gated on CD45⁺DAPI⁻ cells. The result is a representative of at least two independent experiments. C, Sample flow cytometric profile of GFP signal in intra-islet DCs in radiation bone marrow chimeras is shown. The donor-recipient combinations are indicated on top of each plot. D, Summary of the frequencies of GFP⁺ cells in DC gate (CD45⁺DAPI⁻CD11c⁺ cells) in NOD.Rag2^−/− (n = 4), NOD.Rag2^−/−.MIP-GFP (n = 7), NOD.Rag2^−/− into NOD.Rag2^−/−.MIP-GFP chimeras (n = 4), and NOD.MIP-GFP into NOD.Rag2^−/− chimeras (n = 6). Error bars represent SDs. E, Confocal microscopic analysis of GFP in intra-islet, lung, and splenic DCs in NOD. Rag2^−/−.MIP-GFP transgenic mice. Islets and lung cells were dissociated and stained for CD11c (red). The presence of GFP (green) in CD11c (red)-expressing cells was examined by confocal microscopy (original magnification = ×100, scale bar = 5 µm). Fluorescent images show a single medial Z-plane. Phase-contrast images are shown on top for reference. The result is a representative of five independent experiments.

FIGURE 5

Phenotype of intra-islet DCs in NOD.MIP-GFP mice. Single-cell suspension of islets isolated from NOD.MIP-GFP or NOD.MIP-GFP. Rag2^−/− was analyzed by flow cytometry. Histogram overlays of GFP fluorescence (A, left) and cell surface expression of MHC class II (B), and CD40 (C) in NOD.MIP-GFP (shaded histograms) vs NOD.MIP-GFP. Rag2^−/− (bold lines) are shown. A summary chart of percentages of GFP⁺ cell DC gate (CD45⁺DAPI⁻CD11c⁺) in the islets of NOD.MIP-GFP (n = 11) and NOD.MIP-GFP.Rag2^−/− (n = 7) mice is shown (A, right). The p value was obtained using unpaired Student’s t test with Welch’s correction.

FIGURE 6

Kinetic changes in DCs in inflamed islets. A, NOD.MIP-GFP.Rag2^−/− mice received CD4⁺CD25⁻ cells from BDC2.5 TCR transgenic mice. GFP fluorescence and cell surface expression of MHC class II, CD40, and CD80 on intra-islet DCs were analyzed by flow cytometry on days 6 and 9 after cell transfer. Examples of a NOD.Rag2^−/− and a NOD.MIP-GFP.Rag2^−/− mouse without T cell transfer are shown for comparison. This result is representative of at least five independent experiments. B, NOD.Yet40.Rag2^−/− mice received CD4⁺CD25⁻ cells from BDC2.5 TCR transgenic mice. On day 8 postcell transfer, the presence of YFP signal among intra-islet DCs was assessed by flow cytometry. An age-matched untreated NOD.Yet40. Rag2^−/− sample is shown for comparison. This result represents two independent experiments. Events shown are gated on CD45⁺DAPI⁻CD11c⁺ cells in islets.

FIGURE 7

Increase of DC numbers in inflamed islets. A, Islets were isolated from untreated NOD.CD11c-YFP.Rag2^−/− mice (left) or from similar mice 8 days after transferring CD4⁺CD25⁻ cells from BDC.2.5 TCR transgenic mice (right) were counterstained with CMTMR (shown in blue) and imaged on a two-photon microscope. The images are collages of consecutive objective fields representing imaging volume of 780 × 618 × 176 µm³ for untreated islets (left) and 780 × 618 × 222 µm³ for inflamed islets (right). B, Islets were isolated from untreated NOD.Rag2^−/− mice and similar mice 6–7 days after receiving CD4⁺ CD25⁻ cells from BDC2.5 TCR transgenic mice. Expression of Ki67 by intra-islet DCs was analyzed by flow cytometry. Sample contour plots of anti-Ki67 staining and isotype control Ab staining patterns are shown, and a chart summarizing results from two independent experiments is shown below. Events shown are gated on CD45⁺ CD11c⁺ cells. Each circle represents one islet, and the lines represent mean of the group.

FIGURE 8

Phenotypic changes in intra-islet DCs in B6.MIP-GFP.RIP-mOVA mice after inflammation was induced. B6.MIP-GFP.RIP-mOVA mice received CD8⁺ cells from OT-I TCR transgenic mice. GFP fluorescent and cell surface expression of CD40 on intra-islet DCs were analyzed by flow cytometry on day 5 after cell transfer and compared with those from untreated B6 and B6.MIP-GFP.RIP-mOVA mice. Representative dot plots are shown on top, and a summary of all mice analyzed is shown in the chart on the bottom. Values of p were obtained using a one-way ANOVA test with Tukey’s post-test. The result represents four independent experiments.

FIGURE 9

Migration of intra-islet DCs to the PLNs. A, Flow cytometric analysis of GFP signal in DCs in the PLNs of B6.MIP-GFP (left) or NOD.MIP-GFP.Rag2^−/− (right) mice. Events shown are gated on CD45⁺DAPI⁻CD11c⁺ cells. B, Flow cytometric analysis of GFP⁺ DCs in the PLNs of NOD.MIP-GFP mice (left), NOD.MIP-GFP mice 5 days after injection of activated CD4⁺CD25⁻ cells from BDC.2.5 TCR transgenic mice (middle), and 7-wk-old NOD.MIP-GFP.CD28^−/− mice (right). Results are representative of at least two independent experiments. C, Flow cytometric analysis of CCR7 expression on intra-islet DCs in prediabetic NOD.MIP-GFP mice. Contour plots show lymphocytic infiltration into the islets of representative early prediabetic mice (10 wk) vs late prediabetic mice (16 wk), and are gated on CD45⁺DAPI⁻ cells. The histogram overlay (top) shows CCR7 expression in the CD45⁺DAPI⁻CD11c⁺ population from the early vs late prediabetic islets. Histogram overlays (below) show GFP fluorescence and CD40 expression in the CCR7⁺ vs CCR7⁻ populations of intra-islet DCs from late prediabetic mice.