IDO protein and mRNA expression in Ad-IDO transfected cells.

<p>Dermal fibroblasts from C57BL/6 (solid bars) and NOD (open bars) mice were transduced with Ad-IDO or mock vector. <b>A</b>: IDO expression was analyzed by western blotting, <b>C</b>: IDO expression was analyzed by RT-PCR. <b>B</b> and <b>D</b>: the Mean±SEM ratio of IDO to β-actin at the protein and GAPDH at mRNA level (n = 3). β-actin and GAPDH were used as a loading control for protein and mRNA expression respectively. ND: not detected.</p

Different effect of IFN-γ on IDO expression in dermal fibroblasts of C57BL/6 prediabetic NOD mice.

<p>Dermal fibroblasts from prediabetic (8 weeks of age) male and female NOD mice failed to respond to IFN-γ induced IDO. Dermal fibroblasts isolated from C57BL/6 male mice of 8 weeks of age as control (solid bars), and aged matched male (hatched bars) or female (open bars) prediabetic NOD mice were treated with 1000 U/ml of IFN-γ for 48 hours. <b>A</b>: Kyn levels in CM of treated cells, <b>B</b>: IDO expression at the protein level, <b>C</b>: the Mean±SEM ratio of densities of IDO to β-actin at protein control group treated with IFN-γ (n = 3, p<0.01). β-actin expression showed equal loading of proteins. ND: not detected.</p

IFN-γ-induced-STAT1 phosphorylation in C57BL/6 and NOD dermal fibroblasts.

<p>Following starvation for 18 hours, dermal fibroblasts from NOD (open bars) and C57BL/6 (solid bars) mice were remained untreated or treated with 1000 U IFN-γ per ml of DMEM plus 2% FBS for 15, 30 or 60 minutes. Cell lysates were collected for western blot analysis. <b>A</b>: STAT 1 phosphorylation shown by western blotting. <b>B</b>: the Mean±SEM ratio of phospho-STAT1 (P-STAT1), to the ratio of β-actin to total STAT1. Total STAT1 and β-actin expressions were used as loading controls. *denotes significant difference between related bars (p<0.05, n = 3). UT: untreated, ND: not detected.</p

MHC-I mRNA expression in fibroblasts isolated from control and NOD mice.

<p>Cells were treated with 0 or 1000 U/ml of IFN-γ for 48 hours. <b>A</b>: RT-PCR analysis of MHC-I mRNA expression. <b>B</b>: the Mean±SEM ratio of densities of MHC-I to GAPDH. Solid and open bars represent C57BL/6 and NOD fibroblasts respectively. GAPDH was used as loading control. *denotes significant difference between C57BL/6 and NOD fibroblasts treated with IFN-γ in terms of MHC-I expression (n = 3, p<0.05). **corresponds to significant difference between cells from the same strain treated with 0 or 1000 U/ml of IFN-γ (n = 3, p<0.01).</p

COL-I expression in dermal fibroblasts from control and NOD mice.

<p>COL-I expression in dermal fibroblasts from C57BL/6 (solid bars) and NOD (open bars) mice was evaluated by western blot and RT-PCR analyses. Cells were exposed to 0 or 1000 U/ml of IFN-γ for 48 hours before analysis. <b>A</b>: COL-1 expression at the protein level. <b>C</b>: COL-1 expression at mRNA level. <b>B</b> and <b>D</b> represent the Mean±SEM ratio of COL-1 to β-actin at protein and mRNA levels respectively. β-actin was used as loading control in both western blotting and RT-PCR assays. *demonstrates significant difference between C57BL/6 and NOD fibroblasts treated with IFN-γ in terms of COL-1 expression. **corresponds to significant difference between cells from the same strain treated with 0 or 1000 U/ml of IFN-γ (n = 3, p<0.05).</p

Histology of pancreas following cell therapy.

<p>Histology of representative pancreas sections of NOD mice before (at the commencement of autoimmune diabetes) and after cell therapy is shown. Panel <b>A</b> shows hematoxylin-eosin staining (left and center) and insulin immuno-staining (right) of pancreas sections of a NOD mouse euthanized in the first week after the onset of hyperglycemia. Panels <b>B</b> and <b>C</b> show pancreatic sections of an untreated control NOD mouse with chronic diabetes (6 weeks post-diabetes), an IDO cell treated NOD mouse (18 weeks post-treatment), respectively. Middle and right columns show higher magnification (×400) of the marked areas of the left column (×100). Panel <b>D</b> shows Insulitis scores calculated as described in Methods. The ratio (%) of islets with insulitis significantly decreased after IDO cell therapy animals compared to those of the early onset diabetic mice and controls (<b>E</b>). (*) denotes significant decrease in islet insulitis in IDO groups compared to the controls and diabetes onset (P<0.001, <i>n</i> = 5).</p

Increased frequency of CD4⁺ CD25⁺ FoxP3⁺ cells after co-culture with IDO-expressing fibroblast.

<p>Lymphocytes were isolated from mesenteric lymph nodes of recently diabetic NOD mice and were cultured either alone or co-cultured with control or IDO-expressing fibroblasts as described in Methods. The frequency of CD4⁺ gated CD25⁺ FoxP3⁺lypmocytes were measured using flow cytometry before culturing (Pre-culture) and after 7 days mono- or co-cultures. Panel A shows representative plots for CD25+ FoxP3+ regulatory T cells (gated on CD4+ T cells) and panel B shows frequencies of these cells in different experimental groups. (*) denotes statistically significant increase in the frequency of regulatory T cell after co-culturing with IDO-expressing fibroblasts compared to pre-culture amount (P<0.001, n = 3).</p

Expression of CC-chemokine receptor 7 (CCR7) by dermal fibroblasts.

<p>IDO-expressing and control mouse dermal fibroblasts were examined for expression of CCR7 before and after intraperitoneal injection. To track them, fibroblasts were labeled with PKH26 red fluorescent cell membrane labeling kit. Panel <b>A</b> shows representative flow cytometry plots showing frequency of CCR7⁺ cells gated on PKH26⁺ CD90⁺ window. Upper and bottom rows show IDO-expressing and control fibroblasts data, respectively. The plots on the left side show fibroblasts before peritoneal injection (Before inj.). The middle plots and right side plots show cells that were harvested from peritoneal cavity by lavage (Perit. Lav.) or extracted from mesenteric lymph nodes (Mesent. Ln.) two weeks post IP injection, respectively. Panel <b>B</b> show quantification of CCR7 expression on IDO-expressing and control fibroblasts before and after IP injection in peritoneal cavity and mesentric lymph nodes. (*) denotes statistically significant difference in CCR7 expression on IDO and control fibroblasts following IP injection mice compared to the before injection level (P<0.0001, <i>n</i> = 5).</p

Frequency of regulatory, autoreactive CD8⁺, and Th₁₇ T cells in NOD mice after cell therapy.

<p>NOD mice received IDO-expressing fibroblasts upon initiation of spontaneous diabetes. At the endpoint of the study, spleens and pancreatic lymph nodes of these mice were harvested and frequencies of various subtypes of T cells were measured using flow cytometry. Panels <b>A</b> & <b>B</b> show the frequencies of CD 25⁺ FoxP3⁺ regulatory T cells (gated on CD4⁺ T cells) in spleens (SPL) and pancreatic lymph nodes (PLN) of mice, respectively. Autoreactive T cells were detected using NRP-V7 high-avidity peptide/MHC class I tetramers as described in Methods. Panels <b>C</b> &<b>D</b> show representative plots for CD8⁺ tetramer positive cells in spleen (SPL) and pancreatic lymph nodes (PLN) using a lymphocyte gate and excluding CD4⁺ and B220⁺ cells. The numbers in upper right corner of plots indicate the percentage of CD8⁺ B220^- CD4^- tetramer⁺ cells. Panels <b>F</b> & <b>G</b> show representative plots for IL-17 and CD4 flow cytometry analysis of lymphocytes in spleens (SPL) and pancreatic lymph nodes (PLN). The numbers in upper right corner of plots indicate the percentage of CD4⁺ IL-17⁺ (Th₁₇) cells. Panels <b>E</b> & <b>H</b> show quantification of the frequencies of autorective CD8⁺ T cells and Th₁₇ cells, respectively. (*) denotes significant decrease of inflammatory cells in IDO groups compared to the controls (P<0.001, <i>n</i> = 5).</p

Reversal of hyperglycemia in NOD mice after cell therapy.

<p>IDO-expressing or control fibroblasts were injected intraperitoneally to NOD mice with overt hyperglycemia in the first two weeks following the onset of diabetes. These mice did not receive any other treatment. Blood glucose levels were checked twice a week. Panels <b>A</b> show blood glucose levels in IDO treated mice (<i>n</i> = 11 mice). Time point 0 denotes fibroblasts injection time. All control animals (<b>B</b>) that received no cells, control fibroblasts (Con. Fib.) or IDO expressing fibroblasts and an IDO inhibitor, 1-methyl tryptophan (IDO Fib. + 1MT) remained hyperglycemic (<i>n</i> = 5 mice/ group). Kaplan-Meier survival curve with log-rank analysis (<b>C</b>) showed significant decrease in diabetes rate of IDO fibroblast treated group. Intraperitoneal glucose tolerance tests (<b>D</b>) showed improvement of glycemic control in the IDO cell therapy group (red squares) compared to the control mice (blue diamonds).</p