Table 1.
Induction of diabetes in P14/RIP-gp and RIP-gp mice using peptide/adjuvant vaccines.
Figure 1.
Peptide vaccination with anti-CD40 and LPS promotes CD8+ cell expansion and infiltration but not diabetes.
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). *P = 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.
Figure 2.
Transfer of mature DC induces diabetes in RIP-gp mice.
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.
Figure 3.
DC transfer induces CD8 and CD4 function in vivo.
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.
Figure 4.
DC transfer promotes enhanced CTL functions compared to peptide plus adjuvant vaccination.
Total splenocytes were isolated on day 6 following vaccination with unstimulated DC loaded with peptides gp33–41, gp276–286, and gp61–80 (unstim DC), CpG-matured, peptide-loaded DC (CpG DC), or peptides mixed with LPS and anti-CD40 (LPS+anti-CD40), as well as naive (unvaccinated) and LCMV infected controls. (A) IFN-γ and TNF-α expression in CD8+ T-cells were measured following peptide restimulation. (B) Splenocytes were stained with anti-CD8 antibody and gp33–41 or gp-34–41 tetramers. (C) gp33–41 and control AV peptide pulsed and CFSE labeled target cells were transfer into vaccinated mice on day 6 post immunization. 4 hrs later, splenocytes were harvested and examined by flow cytometry for specific lysis of targets. Results show means+/−S.D. from 3 mice per group and are representative of 3–5 independent experiments. **P<0.01; *P<0.05.
Figure 5.
Activation of T-cells specific for at least 2 different peptides is required for diabetes induction.
(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. *P<0.02. Results are representative of 10–15 mice per group.