Long-Term Remission of Diabetes in NOD Mice Is Induced by Nondepleting Anti-CD4 and Anti-CD8 Antibodies
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Figures
- FIG. 1.
Short-course treatment with YTS177 and YTS105 rapidly induces remission in recent-onset diabetic NOD mice that is maintained long-term. A: Recent-onset diabetic NOD female mice were treated with YTS177 and YTS105 (n = 24; top panel) or control 2A3 (n = 5; bottom panel) and blood glucose was monitored. B: Blood glucose levels of individual recent-onset diabetic NOD mice prior to and 72 h after YTS treatment. *P < 10−3. C: Recent-onset diabetic NOD female mice were treated with only YTS105 (n = 5; top panel) or YTS177 (n = 8; bottom panel) and blood glucose was monitored. D: Blood glucose levels were measured in individual 12–14-week-old prediabetic (n = 17) or YTS-treated, long-term remission (≥250 days; n = 18) NOD female mice (left panel) *P < 10−3. Tolerance to injected glucose was assessed in groups of three to six animals (right panel); YTS-treated NOD female mice remaining free of recurrent diabetes ≥150 days were tested.
- FIG. 2.
YTS177 and YTS105 treatment induces T-cell loss in the pancreas and PLN. A: IL-2 and IFN-γ were measured via ELISA in pancreatic homogenates prepared from groups of recent-onset diabetic NOD mice treated with YTS177 and YTS105 (YTS), control 2A3 (Ctrl), or left untreated (Diabetic). Data are the average of eight individual mice. *P ≤ 0.008. The number of CD4+ and CD8+ T cells in the pancreas (Pan), PLN, and spleen (Spl) of NOD (*P ≤ 0.02) (B), NOD.BDC.2.5 (*P < 10−3) (C), and NOD.8.3 (*P ≤ 0.037) (D) was determined via FACS 6 days post-YTS or control 2A3 treatment. Data are the average of 8–15 individual mice. E: The number (left panel) and frequency (right panel) of Foxp3+ Treg in the respective tissues was determined in groups of six NOD.BDC.Foxp3.GFP female mice. *P ≤ 0.01.
- FIG. 3.
YTS177- and YTS105-induced T-cell loss in the pancreas and PLN is not due to apoptosis. A: Representative FACS histograms for TUNEL staining of CD8+ thymocytes in NOD mice 12 h postdexamethasone (Dex) treatment and CD4+ and CD8+ T cells in the PLN of NOD.BDC2.5 and NOD.8.3 mice, respectively, treated with YTS or control 2A3. B: The frequency of TUNEL staining T cells in PLN of individual YTS and isotype control mAb-treated NOD.BDC2.5 and NOD.8.3 mice over time. C: Representative immunofluorescence of thymic sections from control and Dex-treated NOD mice or pancreatic sections from YTS versus control 2A3-treated NOD.BDC2.5 (36 h) and NOD.8.3 (24 h) mice stained with TUNEL (FITC), anti-CD90.2 (PE), and anti-insulin (Alexa 647). (A high-quality color representation of this figure is available in the online issue.)
- FIG. 4.
TGF-β is necessary for YTS177- and YTS105-induced diabetes reversal. A: TGF-β was measured in individual pancreases from groups of three recent-onset diabetic NOD mice treated with YTS, control 2A3, or left untreated and from unmanipulated B6 mice (left panel; *P ≤ 0.0017) and serum at the time of onset and post-YTS treatment in five remission NOD female mice (right panel). The data are representative of at least three experiments. B: Recent-onset diabetic NOD female mice were treated with YTS177 and YTS105 plus anti–TGF-β (n = 9; left panel) or isotype control mAb (n = 10; right panel) and blood glucose was monitored. C: Recent-onset diabetic NOD (n = 7; left panel) and NOD.IL4null (n = 8; right panel) female mice were treated with YTS plus anti–IL-10 receptor mAb or YTS alone, respectively, and blood glucose was monitored. D: NOD.BDC2.5/Cα−/− mice were treated with YTS177 plus anti–TGF-β or isotype control mAb, and 6 days later, the number of CD4+ T cells in the pancreas determined via FACS. *P < 10−3. E: Fold increase in TGF-β1 mRNA in DC, macrophages (MΦ), and T cells sorted from handpicked islets and PLN of individual NOD.BDC2.5 mice (n = 4 to 5/group) 6 days after treatment with YTS or isotype control. DC: *P = 0.004; MΦ: *P = 0.048.
- FIG. 5.
Long-term remission NOD mice have altered β-cell–specific T-cell reactivity in the PLN. Cytokine production by β-cell–specific T cells was measured in the spleen (A) and PLN (B) (*P ≤ 0.04) of YTS-treated animals in remission for >200 days and control recent-onset diabetic NOD mice. Shown is the average of two to four experiments. In medium-only controls, ≤10 IFN-γ or IL-4 spot-forming units (SFU) (left and middle panels) and ≤50 pg/mL of TGF-β (right panel) were detected in enzyme-linked immunospot and ELISA, respectively. C: The frequency (left panel) and number (right panel) of Foxp3+CD25+CD4+ T cells in the spleen (Spl) and PLN of diabetic (Ctrl; n = 6–10) and YTS-treated long-term remission (YTS; >200 days; n = 7–10) NOD female mice as determined by FACS. *P = 0.01.
- FIG. 6.
PLN but not the spleen (Spl) of YTS-treated long-term remission NOD mice contains Treg. Groups of 5–10 NOD.scid mice received diabetogenic splenocytes plus splenocytes (A, left panel) or splenic CD25+CD4+ T cells (A, right panel) and PLN cells (B, left panel) or PLN-derived CD25+CD4+ T cells (B, right panel) isolated from YTS-treated long-term remission (>200 days) or control 2A3-treated diabetic NOD female mice. *P ≤ 0.002. C: Proliferation of CFSE-labeled BDC2.5 CD4+ T cells was measured via FACS (representative histograms provided) in the PLN of YTS Ab-treated long-term remission (>150 days) and nondiabetic 16-week-old NOD female mice. Data are the average of six mice/group. *P = 10−4.
- FIG. 7.
Immunity to foreign antigens is unaffected by YTS Ab treatment. A: Representative FACS plots (left panel) of in vivo CTL activity specific for HA512–520-pulsed targets in nondiabetic untreated (Naive) or peptide-immunized (Ctrl) NOD female mice and YTS Ab-treated long-term remission NOD mice (>150 days) immunized with HA512–520 average specific lysis for groups of eight mice (right panel). B: KLH-specific serum IgM (left panel) and IgG (right panel) were measured in untreated or KLH-immunized NOD female mice or YTS Ab-treated long-term remission NOD mice (>150 days) immunized with KLH.
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