Article Figures & Data
Figures
- FIGURE 1.
Islet Ag-responsive T cells exist in NOD-IL-4. Splenocytes from 8-wk-old female NOD or NOD-IL-4 mice were cultured for 5 days in the presence of GAD65, HSP65, or GAD65 peptides. Proliferation was determined by measuring the amount of [3H]thymidine incorporated (A) over the last 18 h of culture and is expressed as the SI. Culture supernatants were sampled 48 h after Ag administration, and IL-2 levels (B) were measured in a NK-1 bioassay. Each group contained three mice stimulated individually with Ag. Error bars represent the SD. The background levels of lymphocyte proliferation for NOD-IL-4 and NOD mice were 3,470 and 10,327, respectively.
- FIGURE 2.
TCR Vβ T cell repertoire in NOD-IL-4 mice is similar to that in NOD mice. Lymphocytes from the spleen and mesenteric or pancreatic lymph nodes of 7-wk-old female mice were assessed by FACS for CD4 and TCR Vβ expression. Individual mice were evaluated, and results shown are representative of three separate experiments. Vβ 5 represents 5.1 and/or 5.2, and Vβ 8.1 represents 8.1 and/or 8.2.
- FIGURE 3.
Pancreatic IL-4 restores cytokine production to lymphocytes draining the pancreas. Lymphocytes from the pancreatic lymph nodes of three NOD (□) or NOD-IL-4 (•) mice were pooled and stimulated in triplicate with anti-CD3. Culture supernatants were analyzed for proliferation (A), IL-2 (B), IFN-γ (C), and IL-4 (D) after 48 h of stimulation.
- FIGURE 4.
GAD65-specific T cell responses from NOD-IL-4 mice display a Th2 phenotype. Splenocytes from individual female NOD (□) and NOD-IL-4 (•) mice at 5 (A and B) and 8 (C and D) wk of age were cultured with GAD65. Proliferation (A) and IL-2 (C) production were determined as described in Fig. 1. To assess the cytokines produced from GAD65-specific T cells, GAD65 expanded splenocytes were stimulated nonspecifically through the TCR complex using anti-CD3 mAb. Culture supernatants were evaluated for IFN-γ by ELISA (B) and IL-4 (D) in a bioassay at 48 and 72 h, respectively. The number of individual GAD65 expanded T cells producing IL-4 or IFN-γ was determined by ELISPOT (E) and expressed as SFC.
- FIGURE 5.
Intrapancreatic levels of IFN-γ are similar in NOD and NOD-IL-4 mice. The pancreata from five NOD or NOD-IL-4 mice were freshly frozen, homogenized, and assessed for IFN-γ levels by ELISA. Error bars represent the SD.
- FIGURE 6.
Anti-GAD65 Ab responses in NOD-IL-4 mice display a Th2 profile. Sera from five NOD and NOD-IL-4 mice (8–10 wk old) were analyzed individually by ELISA for IgG subclasses specific for GAD65. Error bars represent the SD between individual mice.
Tables
T Cell Hybridoma TCR Vβ Expression Thymidine Uptake No Ag GAD65 NOD-IL-4 1F12 8.1, 8.2 3,768 20,092 1B5 8.1, 8.2 32,089 62,198 3E5 8.1, 8.2 20,822 72,054 1D6 8.1, 8.2 2,624 45,908 3B4 8.1, 8.2 3,728 107,055 3D11 4 22,447 56,550 2B4 5.1, 5.2 11,339 60,023 NOD 6G2 8.1, 8.2 931 107,261 7F7 8.1, 8.2 5,313 32,816 7C4 8.1, 8.2 3,754 25,545 8B9 8.1, 8.2 1,638 36,834 7D1 8.3 10,537 56,361 6G5 8.3 1,110 87,419 6H7 10b 2,865 33,742 8B8 4 7,920 117,082 -
a IL-2 secretion by T cell hybridomas upon 24-h incubation with GAD65. The TCR Vβ expression by the respective hybridoma was determined by FACS.
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Group Donor Splenocytes From Recipients Total No. of Islets/Group Grade of Insulitis (%) Insulitis Score 0 1 2 3 A Diabetic NOD 77 12 39 26 23 1.6 Diabetic NOD-IL-4 48 65 29 6 0 0.41b B GAD65-stimulated NOD 75 15 12 32 41 2.0 GAD65-stimulated NOD-IL-4 89 69 19 11 1 0.45b C Diabetic+ NOD NOD.scid 48 15 29 45 11 1.5 Diabetic+ NOD-IL-4 NOD-scid 88 48 33 19 0 0.71** D GAD65-stimulated-NOD+ diabetic NOD.scid 41 18 24 35 23 1.6 GAD65-stimulated-NOD-IL-4+ diabetic NOD.scid 47 69 19 13 0 0.45** -
a Pancreatic islets were scored for insulitis by hematoxylin and eosin staining 7 days following adoptive transfer of 5 × 106 recently diabetic NOD splenocytes (group A); 1.5 × 107 GAD65 stimulated NOD splenocytes (group B); 3 × 106 diabetic plus 1.5 × 107 NOD or NOD-IL-4 (group C); or 1 × 107 GAD65 stimulated plus 5 × 106 recently diabetic NOD splenocytes (group D).
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b p < 0.0001; ** p < 0.01 (χ2 test).
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- Table III.
Th2 cytokines are responsible for regulating diabetogenic T cells in NOD-IL-4 micea
Donor Splenocytes Ab Treatment Incidence of Diabetes (at wks) Nondiabetic Diabetic 6 7 8 9 10 NOD-IL-4 — Control 0 /3 0 /3 0 /3 0 /3 0 /3 NOD-IL-4 — α-IL-4 0 /3 0 /3 0 /3 0 /3 0 /3 NOD-IL-4 — α-IL-4/α-IL-10 0 /3 1 /3 1 /3 3 /3 — NOD NOD Control 2 /7 5 /7 7 /7 — — NOD-IL-4 NOD Control 0 /4 0 /4 3 /4 3 /4 4 /4b NOD-IL-4 NOD α-IL-4 0 /5 3 /5 5 /5 — — NOD-IL-4 NOD α-IL-4/α-IL-10 1 /4 2 /4 4 /4 — — -
a Splenocytes (15 × 106) from 8 to 10-wk-old nondiabetic NOD-IL-4 or NOD mice were adoptively transferred into NOD.scid mice, with or without splenocytes (3 × 106) from recently diabetic NOD mice. Groups of mice were treated every other day for 2 wk following transfer with α-IL-4 (11B11) and/or α-IL-10 (JES2A5) or control IgG Ab. Mice were scored as diabetic when blood glucose values reached greater than 300 mg/dL. The anti-IL-4 mAb (11B11) were found effective in transplantation model (30) and anti-IL-10mAb (JES2A5) was found effective in autoimmune diabetes (50).
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b p < 0.05 (log-rank test).
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