PMC

Comparison of ex vivo T cells from NOD and Bg7 mice. Thymus and spleen cells were isolated and stained for CD4, CD8, CD25, Foxp3, and TCR-β-chain. Percentage of Foxp3⁺ Tregs per CD4SP was analyzed. (A) Thymi from NOD mice (open symbols), NOD.Ea16 mice (gray symbols, dashed line), and Bg7 mice (black symbols) were isolated at different days after birth, and percentage of Foxp3⁺ per CD4SP T cells was analyzed. Mean and SD of 3–11 mice per group and time point are shown. (B) Broad screen of Tregs among inbred mouse strains. Thymus and spleen from 10 inbred mouse strains were analyzed. Mean percentage of CD4⁺Foxp3⁺ Tregs and SD are shown. Three mice per strain were analyzed. Mean values from spleen and thymus were plotted, and NOD and B6 mice are highlighted.

Differences in peptide-induced and naturally selected Tregs. BDC2.5/NOD and NOD FTOCs were cultured in parallel for 2–7 days. BDC2.5/NOD lobes were supplemented with 10 ng of BDC-specific peptide. (A) Cells were gated for CD4⁺CD8⁻. Bold numbers show percentage of Foxp3⁺ and CD25⁺ or CD25⁻ Tregs. *, Ratio of CD25⁺/CD25⁻ of Foxp3⁺ T cells. Three to four FTOCs per day and group were pooled and one representative experiment of two is shown. (B) (Left) BDC2.5/NOD FTOCs were harvested and gated for Foxp3⁺ cells. Percentage of CD4SP and DP are shown. (Right) Percentage of DP among Foxp3⁺ cells. Filled symbols are BDC2.5/NOD FTOCs; open symbols are NOD FTOCs. Representative dot plots and mean and SD of three independent experiments are shown. (C) FTOCs were cultured for 7 days and gated for CD4SP cells. Canonical Treg-specific proteins were stained and shown in bold are the percentage of Foxp3 DP Tregs. Representative dot plots and mean and SD of three experiments are shown.

Treg induction in BDC2.5/NOD FTOC system. BDC2.5/NOD and NOD FTOCs were cultured for 7 days. BDC2.5/NOD lobes were supplemented with BDC-specific mimotope peptide. (A) Induction of Foxp3⁺ Tregs in FTOCs from NOD and BDC2.5/NOD mice either with or without peptide. Cells were gated for CD4⁺CD8⁻. Numbers show the percentage of Foxp3⁺ Tregs per CD4SP. Representative dot plots are shown. (B) BDC2.5/NOD FTOCs were cultured with different peptide concentrations. Proportion and absolute number of Foxp3⁺ Tregs cells per CD4SP are displayed. (C) Total number of CD4SP and DP cells from the cultures described in B are shown as a measure for deletion (survival). Mean and SD of four independent experiments with three to six pooled FTOCs per peptide concentration and experiment are shown.

Strain differences between NOD and Bg7 in Treg induction. FTOCs were cultured for 7 days. FTOCs cultures were performed in parallel with embryos from BDC2.5/NOD (open symbols) mice and BDC2.5/Bg7 (filled symbols) mice and supplemented with different concentrations of BDC-specific peptide. (A) (Left and Middle) Percentage and absolute number of Foxp3⁺ Tregs per CD4SP. Mean and SD of four independent experiments with each three to six pooled FTOC lobes per concentration and group are shown. (Right) Number at peak shows absolute number of Foxp3⁺ cells at the peptide concentration with a maximum of Treg induction (3 ng for BDC/Bg7 and 30 ng for BDC/NOD). Presented are four individual experiments. (B) Deletion based on percentage of DP cells. (C) Nontransgenic FTOC experiments from NOD mice and Bg7 mice. (Left) Numbers show percentage of Foxp3⁺ Tregs per CD4SP of representative dot plots. (Right) Percentage and absolute number of Foxp3⁺ Tregs per CD4SP and mean and SD of three independent experiments with three to five FTOCs per group and experiment are shown.

Two genomic regions promote an enhanced selection of Tregs in NOD. (A) Repopulation thymus organ culture revealed that differences in Treg selection were intrinsic to the developing T cells. DN thymocytes from E15.5 embryos from the two strains were repopulated into deoxyguanosine-treated FTOC lobes from NOD or Bg7 mice. Donor cells could be discriminated by differences in expression of CD45 (NOD, CD45.1; Bg7, CD45.2). Numbers show the percentage of Foxp3⁺ Tregs per CD4SP. One of two independent experiments is shown. (B) FTOCs from NOD.idd3^b/b, NOD.idd3^b/n congenic, and NOD, Bg7, and F₁ (NODxBg7) mice are shown. Representative dot plots from three to five FTOCs are shown. (C) NOD, Bg7, F₁, and F₂ FTOCs were cultured for 7 days, and expression of Foxp3⁺CD25⁺ Tregs per CD4SP was measured. (D) Logarithm of odds plots for the FTOC/F2 cohorts are shown. Dotted line represents level of significance (P < 0.05). (E) Linkage maps of Chr1 (Left) and Chr11 (Right).