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

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

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