Newly diagnosed diabetic NOD mice were treated as indicated, and glycemia was monitored until 14 weeks after treatment initiation. (A) Percentage of mice that remained diabetic after treatment. † indicates dead or moribund mice. (B) Individual blood glucose levels of newly diagnosed diabetic NOD mice treated with anti-CD3 monotherapy (left panel) or anti-CD3 and LL-PINS+hIL10 CT (right panel). Open symbols, hyperglycemic mice; filled symbols, normoglycemic mice at week 14. (C) Newly diagnosed diabetic NOD mice were stratified based on initial blood glucose level less than (gray symbols) or greater than (open symbols) 350 mg/dl. Shown is the percentage of mice that remained diabetic upon treatment with anti-CD3 monotherapy (left panel) or anti-CD3 and LL-PINS+hIL10 CT (right panel). In all panels, statistical significance between groups was determined by Mantel-Cox log-rank test; *P < 0.05, **P < 0.01.

(A) Evaluation by ELISA of insulin content in pancreatic extracts from cured (14 weeks) and control experimental groups, as indicated. Data are expressed as ng/mg of pancreas. Shown are the individual values (symbols) overlaid with mean (horizontal line), box (25th and 75th percentile), and Tukey whiskers. Statistical significance between 2 groups was calculated using Mann-Whitney U test; *P < 0.05, **P < 0.01. (B) Pancreatic sections were stained for insulin (green), Ki-67 (red), and DNA by Hoechst (blue) and analyzed with confocal microscopy at ×63 original magnification. Representative Ki-67/insulin microphotographs documenting the absence of proliferating β cells in the pancreas are shown. (C) Pancreatic sections of the indicated groups were stained with H&E, and insulitis was scored as indicated.

(A) CD25⁺Foxp3⁺ cells in PLNs, shown as mean ± sem within the CD4 gate. (B) Histogram overlays of FR4 (top) and CTLA4 (bottom) by CD4⁺CD25⁺Foxp3⁺ cells isolated from PLNs (end of treatment). Inset values: MFI of FR4 expression and percentage of CTLA4⁺ cells in CD4⁺CD25⁺Foxp3⁺ gate, respectively. Gray histograms: staining on indicated sample; solid lines: isotype staining on nondiabetic control sample. (C–E) In vitro polyclonal suppressor assay. CD4⁺CD25^– Tresps isolated from normoglycemic NOD mice were dye labeled and stimulated using 0.5 μg/ml soluble anti-CD3 and accessory cells for 72 hours in the presence of CD4⁺CD25⁺ Tregs isolated from cured mice at the end of the indicated treatment. Results of assays for in vitro suppressive capacity are shown. (C) Proliferation of Tresps, shown as percentage of Tresps that had undergone 2 or more divisions, normalized to Tresp-only culture. (D) Activation of Tresps, shown as MFI of CD44 expression, normalized to Tresp-only culture. (E) ELISA for IL-10. (F) Assay for in vivo suppressive capacity. CD25-depleted splenocytes from diabetic NOD mice were transferred into NOD/SCID mice without (white circles) or with CD4⁺CD25⁺ cells from CT-cured (white squares) or CT-non-cured (black squares) mice. Shown is the diabetes incidence in recipients. Statistical calculation using Mann-Whitney test (^#P < 0.05, ^##P < 0.01 versus week 6 CT; ^†P < 0.05, ^††P < 0.01, ^†††P < 0.001 versus week 14 CT), t test (C–E), and Mantel-Cox log-rank (F). Results in C–E are representative of at least 3 experiments (each at least 3 treated mice). *P < 0.05, ***P < 0.001.

(A) Real-time PCR analysis of indicated mRNA isolated from CD4⁺CD25⁺ T cells sorted from spleen and PLN of long-standing normoglycemic, diabetic, and CT-cured NOD mice. (B) Number of Foxp3-expressing cells in or around the pancreatic islets as determined by manual counting of Foxp3⁺ cells on immunostained cryosections as shown in Supplemental Figure 9. (C) Representative microphotographs of immunostaining for Ki-67 and Foxp3 and nuclear staining using DAPI on paraffin sections of the pancreas of CT-cured NOD mice. White arrows indicate Ki-67⁺Foxp3⁺ double-positive cells, indicating proliferating Foxp3⁺ Tregs. Original magnification, ×63; detail, ×252. Statistical significance was calculated using Mann-Whitney U test (*P < 0.05, **P < 0.01, ***P < 0.001).

Cell Proliferation Dye eFluor–labeled (eBioscience) CD4⁺CD25^– Tresps, isolated from normoglycemic NOD mice that had received a prime-boost with human PINS or chicken OVA, were cocultured for 4 days with activated PINS- or OVA-pulsed APCs and the indicated ratios of CD4⁺CD25⁺ T cells, isolated from CT-cured mice at the end of the 6-week treatment. (A) Activation of Tresps was measured by flow cytometric analysis of CD44 and shown as MFI normalized to responder-only culture. (B) ELISA measurement of IFN-γ in the supernatant of Treg-Tresp cultures. (C) Percentage of IFN-γ–positive cells in CD4⁺ Tresps in Treg-Tresp cocultures stimulated with PINS or OVA as indicated. (D) Percentage of IL-10–producing cells in CD4⁺ Tregs in Treg-Tresp cocultures stimulated with PINS or OVA as indicated. Statistical significance was calculated using Mann-Whitney t test (*P < 0.05, **P < 0.01, ***P < 0.001).