Figure 1.
Flow analysis of CD4+CD62L+ Tregs following in vitro co-culture with human cord blood stem cells CB-SC.
Mouse spleen lymphocytes were isolated from female NOD mice (aged 6–8 weeks) and then co-cultured with CB-SC for 2–4 days at different ratios (A) or a ratio 1∶10 of CB-SC∶lymphocytes (B–D). Subsequently, the total lymphocytes were harvested for following cell count or flow analyses. Lymphocytes cultured in absence of CB-SC served as control. (A) CB-SC have no significant effect on mouse lymphocyte proliferation during co-culture at different ratios of CB-SC∶lymphocytes. Data represent mean±s.d. of four experiments. (B) Percentage of CD4+CD25+ Treg, CD4+Foxp3+ Treg, and CD4+CD62L+ Treg after in vitro co-culture with CB-SC. (C) Flow analysis of CD25 and Foxp3 expressions in CD4+CD62L+ Tregs after in vitro co-culture with CB-SC. (D) Flow analysis on CD4+CD62L+ Tregs after intra-cellular cytokine staining. Isotype-matched IgG served as control. Data in B–C are representative of three to five experiments.
Figure 2.
CB-SC-modulated CD4+CD62L+ Tregs (mCD4CD62L Tregs) reverse hyperglycemia in overt diabetic NOD mice.
Mouse spleen lymphocytes isolated from female NOD mice (aged 6–8 weeks) were co-cultured with CB-SC for cell sorting as described in methods. These purified CD4+CD62L+ Tregs (mCD4CD62L Tregs) were used for treatment of spontaneously-developed autoimmune-caused diabetes in NOD mice. The purified CD4+CD62L+ Tregs from lymphocytes not co-cultured with CB-SC (control CD4CD62L Tregs) served as control for mCD4CD62L Tregs. Injection of vehicle PBS served as an additional control (n = 5). (A) The mCD4CD62L Tregs correct hyperglycemia in diabetic NOD mice. Overt diabetic NOD mice were treated with mCD4CD62L Tregs (total 5 million cells/mouse, i.p., red line; representative data are from 6 diabetic mice sensitive to mCD4CD62L Treg treatment with euglycemia, n = 8 mice). Purified control CD4CD62L Tregs served as control (total 5 million cells/mouse, i.p., blue line, n = 5 mice). PBS served as an additional control (black line, n = 5 mice). (B) Intraperitoneal glucose tolerance testing (IPGTT) 3 weeks following the 1st treatment with mCD4CD62L Tregs. Seven-week old NOD mice served as normal control. (C) Determination of blood insulin levels by ELISA. (D) Effect of treatment on mouse body weight. (E–H) Pancreatic histology analyses: Pancreata from mCD4CD62L Treg-treated diabetic mice and control mice were collected for immunohistochemistry after observation for 45 days. Representative sections are shown in each panel (G, H). (E) Morphometric analysis of pancreatic β-cell mass. Pancreatic β-cell mass was determined by point-counting morphometry on insulin-positive islet β cells followed by immunostaining with guinea pig anti-insulin Ab (Dako) and counter-staining with hematoxylin. (F) Quantification of Ki67-positive cells in pancreatic islets after double immunostaining with Ki67 and insulin Abs. Isotype-matched rabbit IgG served as control for rabbit anti-Ki67 mAb. (G) Confocal microscopy shows double-immunostaining for insulin (red) and a cell proliferation nuclear marker Ki67 (green) (scale bar, 50 µm), with a high magnification (two bottom rows, scale bar 10 µm). Control CD4CD62L Treg-treated diabetic mice (top panels) showed the Ki67-positive cells distributed in the infiltrated inflammatory cells (blue, with high density), not in β-cell area (dashed pink circle), with almost complete disappearance of β cells (red). (H) Double-immunostaining for β-cell marker insulin (red) and α-cell marker glucagon (green), followed by nuclear counter-staining with DAPI (blue). Seven-week old NOD mice served as non-diabetic control to show normal islet architecture. Scale bar, 50 µm.
Figure 3.
Treatment with mCD4CD62L Tregs reverses insulitis and immune dysfunction in diabetic NOD mice.
Overt diabetic NOD mice treated with mCD4CD62L Tregs were sacrificed for pancreatic histological analysis and evaluation of blood cytokine levels after observation for 45 days (n = 8). The control CD4CD62L Treg-treated diabetic mice (n = 5) and PBS-treated diabetic mice (n = 5) served as controls. (A and B) Treatment with mCD4CD62L Tregs corrects insulitis in overt type 1 diabetic NOD mice. Representative data are from 6 diabetic mice (6/8 mice) sensitive to mCD4CD62L Treg treatment with euglycemia. (A) Scoring of insulitis. Pancreatic islets were scored for % mononuclear cell infiltration after immunostaining for insulin and counter-staining with hematoxylin as described in methods. (B) Representative images for different type of insulitis. Data were collected from mCD4CD62L Treg-treated diabetic NOD mice. Scale bar, 50 µm. (C) Determination of plasma IFN-γ level by ELISA. Non-diabetic NOD mice at age of 6 weeks served as normal control. (D) Measurement of plasma IL-4 level by ELISA. (E) Determination of plasma IL-10 level measured by ELISA. (F) Determination of plasma TGF-β1 level measured by ELISA. Data are shown as mean±s.d. of mouse plasma cytokine levels from three experiments.
Figure 4.
Treatment with mCD4CD62L Tregs enhance expression of TGF-β1 in pancreatic islets.
Overt diabetic NOD mice treated with mCD4CD62L Tregs were sacrificed for pancreatic immunohistochemistry studies after observation for 45 days (n = 8). Representative data are from 6 diabetic mice (6/8 mice) sensitive to mCD4CD62L Treg treatment with euglycemia. The control CD4CD62L Treg-treated diabetic mice served as control (n = 5). (A) TGF-β1 staining surrounds a pancreatic islet of mCD4CD62L Treg-treated diabetic mice, as determined by double-immunostaining for TGF-β1 (yellow) and insulin (red). TGF-β1 positive cells (bright yellow) and released TGF-β1 in matrix (faint yellow) were distributed in the islet β cell area (red) and surrounded islet β cells (top panels, scale bar 50 µm). High magnification is shown in bottom panels, scale bar 10 µm. Isotype-matched mouse IgG1 served as a negative control for TGF-β1 immunostaining in a serial pancreatic section. Representative images were obtained from five experiments. (B) TUNEL assay. The proteinase K-pretreated pancreatic slides were initially immunostained with TGF-β1 (yellow) and insulin (red) Abs, followed by TUNEL assay (green), and nuclear counterstaining with DAPI (blue). Scale bar 20 µm. Representative images were obtained from four experiments. (C) Percentage of apoptotic cells in subtypes of infiltrated leukocytes in pancreatic islets. Cryosections (8 µm thickness) of frozen pancreata from mCD4CD62L Treg-treated diabetic mice (n = 4) and control mice (n = 4) were initially detected with In Situ Cell Death Detection Kit (Roche), followed by immunostaining with different monoclonal Abs and imaging with a Zeiss LSM 510 META confocal microscope. Cryosections incubated with label solution without the TUNEL reaction mixture and/or isotype-matched IgG served as negative controls. Data represent mean±s.d. of five experiments.