Limited CD8 infiltration in mice treated with LPS matured DCs.

<p>Degree of CD8+ islet infiltration in RIP-gp mice treated with LPS stimulated peptide-pulsed p40+/+ or p40−/− BMDCs. Results are from a minimum of 5 mice per group, 100 islets per group from 2 independent experiments.</p

APC stimulation confers differential requirement of IL-12 in the induction of autoimmunity.

<p>(A) Diabetes incidence in RIP-gp/p40+/+ (solid circle) and RIP-gp/p40−/− (solid square) mice that were infected with LCMV-WE on day 0. Results are from 9 mice per group, 2 independent experiments. (B, C) Diabetes incidence in RIP-gp/p40+/+ (open circle) and RIP-gp/p40−/− (open triangle) mice treated with p40+/+ and p40−/− BMDCs respectively, pulsed with peptides derived from LCMV-GP and stimulated with (B) LPS or (C) Poly I∶C. (D) Diabetes incidence in RIP-gp/p40+/+ (solid square) and RIP-gp/p40−/− (solid diamond) mice treated with p40−/− and p40+/+ BMDCs respectively, pulsed with peptides derived from LCMV-GP and stimulated with LPS. (E) Diabetes incidence in RIP-gp mice treated with IL-12p35−/− (open diamond) and IL-12p19−/− (open square) BMDCs pulsed with peptides derived from LCMV-GP and stimulated with LPS. In each experiment, non-peptide pulsed DCs were included as negative controls (Cross). Results in (B) to (E) are from 7 to 11 mice per group, 2 independent experiments. A test of statistical significance of p<0.05 by the Mantel-Cox Test and Gehan-Breslow-Wilcoxon Test is denoted with * in (B), (D) and (E).</p

Exogenous IFNα enhances CD8 infiltration.

<p>(A) Insulitis as assessed by immunohistochemistry of CD8 infiltrates (stained red) in pancreas sections five days after treatment. Representative sections from RIP-gp mice treated with p40+/+ (bottom left panel), p40−/− (bottom middle panel) peptide-pulsed BMDCs stimulated with LPS or p40−/− peptide-pulsed BMDCs stimulated with LPS with an additional i.v. injection of IFNα (bottom right panel) are displayed. For controls, representative sections of RIP-gp mice treated with unstimulated peptide-pulsed p40+/+ (top left panel) or p40−/− (top middle panel) BMDCs and C57BL/6 mice treated with LPS stimulated peptide-pulsed p40−/− BMDCs (top right panel) are shown. (B) Quantiation of CD8 infiltration, with the first 2 columns represented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0023940#pone-0023940-g002" target="_blank">figure 2</a> but reproduced here for comparison. Results are representative of a minimum of 5 mice per group, 100 islets per group from 2 independent experiments.</p

Exogenous IFNα can overcome the absence of DC-derived IL-12 in the induction of autoimmunity.

<p>(A) IFNα production was measured in the supernatant from unstimulated, LPS stimulated and PolyI∶C stimulated p40+/+ and p40−/− BMDCs by ELISA. Results are from 4 mice per treatment group and representative of 3 independent experiments. (B) Diabetes incidence in RIP-gp mice treated with IFNα alone (open diamond), peptide-pulsed p40−/− BMDCs stimulated with LPS (open square) or peptide-pulsed p40−/− BMDCs stimulated with LPS plus IFNα (open triangle). Injection of 10000 u of IFNα to RIP-gp mice 3 days after treatment with LPS stimulated peptide-pulsed 40−/− BMDCs or LPS and IFNα stimulated peptide-pulsed p40−/− BMDCs (open circle and open upside-down triangle respectively). (C) Diabetes incidence in IFNαR+/+/RIP-gp bone marrow chimeras treated with LPS stimulated p40+/+ BMDCs (open circle) and IFNαR−/−/RIP-gp chimeras treated with LPS stimulated p40−/− BMDCs (open square) or an additional i.v. injection of IFNα 3 days later (open triangle). A test of statistical significance of p<0.05 against RIP-gp mice (B) and IFNαR−/−/RIP-gp mice (C) treated with p40−/− BMDCs stimulated with LPS is denoted with *. Results shown are from a minimum of 6 mice per group from 2 independent experiments.</p

Induction of diabetes in P14/RIP-gp and RIP-gp mice using peptide/adjuvant vaccines.

<p>Mice were infused intravenously with indicated peptides and adjuvants on days 0 and 2 and blood was drawn every 2–3 days from the tail vein to assess blood glucose levels for a minimum of 16 days. Diabetes is defined as a blood glucose of >14 mM on two consecutive readings. Indicated results are for at least 5 mice per group and 2–3 independent experiments.</p

Transfer of mature DC induces diabetes in RIP-gp mice.

<p>Bone marrow derived DCs were generated and cultured overnight in media containing (A) no maturation stimulus, (B) CpG ODN 1826 (10 μM), (C) LPS (10 ng/ml), or (D) imiquimod acetate (25 μM), prior to pulsing with gp33–41, gp276–286, and gp61–80 peptides and tail vein infusion to RIP-gp mice at 2×10⁶ DC/mouse. Blood glucose levels were followed after vaccination. Each line represents an individual mouse. (E) Quantification of diabetes incidence following transfer of DC as in (A-D) for 10–20 mice per group. (F) Quantification of the degree of CD8⁺ and CD4⁺ cell infiltration in pancreatic histology 6 days after transfer of unstimulated or CpG-stimulated peptide-pulsed DCs. All results representative of at least 3 independent experiments.</p

Activation of T-cells specific for at least 2 different peptides is required for diabetes induction.

<p>(A-H) Bone marrow derived DCs were matured with 10 μM CpG ODN 1826 and pulsed with the indicated peptides prior to transfer to RIP-gp mice at 2×10⁶ cells/mouse via tail vein infusion. Blood glucose levels were followed on subsequent days. Each line represents an individual mouse. (I) Quantification of diabetes incidence following transfer of CpG-matured DC pulsed with gp33–41 alone, gp33–41 and gp276–286, or gp33–41 and gp61–80. *<i>P</i><0.02. Results are representative of 10–15 mice per group.</p

Peptide vaccination with anti-CD40 and LPS promotes CD8⁺ cell expansion and infiltration but not diabetes.

<p>Mice were infused intravenously on days 0 and 2 with 10 μg gp33–41, 10 μg gp276–286, 2 μg gp61–80, and 30 μg LPS with or without anti-CD40 agonistic antibody as indicated. (A) Flow cytometry conducted on blood of C57BL/6 mice 7 days after the first vaccination stained using anti-CD8 antibody and gp33–41 tetramer. Representative plots are shown. Naïve and LCMV-infected mice were used as negative and positive controls respectively. (B) Quantification of tetramer-positive populations in blood of C57BL/6 mice after infection with LCMV (triangles) or vaccination with peptides in conjunction with LPS (circles), or LPS and anti-CD40 (squares). *<i>P</i> = 0.0004 (C) Pancreas sections from RIP-gp mice 8 days after receiving peptide vaccine and stained with anti-CD4 or anti-CD8 antibody as shown. (D) Blood glucose measurements from mice vaccinated with peptides, LPS, and anti-CD40 agonistic antibody. Each line represents an individual mouse.</p

DC transfer induces CD8 and CD4 function <i>in vivo</i>.

<p>Bone marrow derived DC were generated and cultured overnight in media with or without CpG ODN 1826 (10 μM) prior to peptide-pulsing and transfer via tail vein infusion at 2×10⁶ cells/mouse. (A) 2×10⁶ CFSE-labeled CD8-sorted P14/Thy1.1⁺ splenocytes were transferred to C57BL/6 mice alone or with a separate infusion of peptide-pulsed CpG-stimulated DCs. 3 days later, splenocytes were isolated and flow cytometry was conducted to determine CFSE dilution of transferred Thy1.1⁺CD8⁺ cells. (B) 2×10⁶ CFSE-labeled CD4-sorted Smarta/Thy1.1⁺ splenocytes were transferred to C57BL/6 mice alone or with a separate infusion of peptide-pulsed CpG-stimulated DCs. 3 days later, splenocytes were isolated and flow cytometry was conducted to determine CFSE dilution of transferred Thy1.1⁺CD4⁺ cells. (C) Quantification of gp33–41-specific production of IFN-γ, TNF-α, and IL-2 by CD8⁺ splenocytes in naïve mice and 8 days after vaccination with unstimulated or CpG-stimulated peptide-pulsed DCs. Results show means+/−S.D. Dot plots are gated on Thy1.1⁺ population. All results are representative of 2–3 independent experiments with >3 mice per group.</p

APC maturation signals promote T cell activation and inflammation, but are unable to induce diabetes.

<p>(a) and (b) CFSE-labeled P14/CD45.1+ T cells were transferred to RIP-iCasp-3.4/gp mice and treated with AP20187. Anti-CD40 antibody was injected 2 days later. After 3 days, cells from the PDLN were analyzed for division by CFSE dilution and IFN−γ production. (c) and (d) CD8 T cell infiltration in the pancreas was evaluated by histology sections which were stained with anti-CD8. (e) Infiltrating P14 T cells and CD8+ T cells in the pancreas were quantified. (f) P14 T cells were transferred into RIP-iCasp-3.4/gp mice. After 24 hours, AP20187 was given i.p., followed by anti-CD40 antibody i.v. 2 days later. Blood glucose levels were monitored every other day for 60 days. At day 49, mice were infected with LCMV-Armstrong. Data are representative of at least 5 mice per condition from independent experiments and error bars in bar graph show S.E.M. (*<i>p</i> < 0.05).</p