Fig. 3.

PLN-derived CD4⁺CD25⁺Treg cells fail to control islet-specific dnTGFβRII CD8⁺ T cells. (A) Tet-TNF-α/CD80 mice were induced to express TNF-α from birth to 25 days of age. On day 32, the PLN-derived CD4⁺CD25⁺ T cells were isolated by using MoFlo. PLN-derived cells from 6-week-old dnTGFβRII mice were incubated with rat anti-B200 and anti-CD4 Abs, and the CD8⁺ T cells were negatively selected by using anti-rat-coated beads. Recipient Tet-TNF-α/CD80 mice were induced to express TNF-α from birth to 28 days of age, and on day 30, groups of mice were injected with 1 × 10⁴ CD4⁺CD25⁺ (♦, n = 3), 1 × 10⁴ CD4⁺CD25⁺, and 3 × 10⁴ CD8⁺ (▪, n = 4), or PBS (▴, n = 4). Diabetes progression was monitored as before. (B) The pancreata were removed from new diabetic recipients of PBS (a), CD4⁺CD25⁺ Treg cell and dnTGFβRII tg CD8⁺ T cell dual transfers (b), or CD4⁺CD25⁺ Treg cells (c), and were fixed in 1% paraformaldehyde. Five- to 7-μm paraffin-embedded sections were incubated with anti-insulin Abs (green) or isotype control Abs (d), and nuclei were stained with DAPI (blue). The data are representative of two individual mice examined from each group.