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. 2010 Sep;59(9):2219-27.
doi: 10.2337/db09-1560. Epub 2010 Jun 3.

Local Expression of Indoleamine 2,3 Dioxygenase in Syngeneic Fibroblasts Significantly Prolongs Survival of an Engineered Three-Dimensional Islet Allograft

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Free PMC article

Local Expression of Indoleamine 2,3 Dioxygenase in Syngeneic Fibroblasts Significantly Prolongs Survival of an Engineered Three-Dimensional Islet Allograft

Reza B Jalili et al. Diabetes. .
Free PMC article

Abstract

Objective: The requirement of systemic immunosuppression after islet transplantation is of significant concern and a major drawback to clinical islet transplantation. Here, we introduce a novel composite three-dimensional islet graft equipped with a local immunosuppressive system that prevents islet allograft rejection without systemic antirejection agents. In this composite graft, expression of indoleamine 2,3 dioxygenase (IDO), a tryptophan-degrading enzyme, in syngeneic fibroblasts provides a low-tryptophan microenvironment within which T-cells cannot proliferate and infiltrate islets.

Research design and methods: Composite three-dimensional islet grafts were engineered by embedding allogeneic mouse islets and adenoviral-transduced IDO-expressing syngeneic fibroblasts within collagen gel matrix. These grafts were then transplanted into renal subcapsular space of streptozotocin diabetic immunocompetent mice. The viability, function, and criteria for graft take were then determined in the graft recipient mice.

Results: IDO-expressing grafts survived significantly longer than controls (41.2 +/- 1.64 vs. 12.9 +/- 0.73 days; P < 0.001) without administration of systemic immunesuppressive agents. Local expression of IDO suppressed effector T-cells at the graft site, induced a Th2 immune response shift, generated an anti-inflammatory cytokine profile, delayed alloantibody production, and increased number of regulatory T-cells in draining lymph nodes, which resulted in antigen-specific impairment of T-cell priming.

Conclusions: Local IDO expression prevents cellular and humoral alloimmune responses against islets and significantly prolongs islet allograft survival without systemic antirejection treatments. This promising finding proves the potent local immunosuppressive activity of IDO in islet allografts and sets the stage for development of a long-lasting nonrejectable islet allograft using stable IDO induction in bystander fibroblasts.

Figures

FIG. 1.
FIG. 1.
Islet graft survival and function after transplantation. A: Kaplan-Meier survival curve shows prolongation of IDO-expressing grafts survival (solid line) compared with islet alone (dash-dot line), untreated (dashed line), and mock virus–infected (dotted line) grafts (n = 10). IPGTT after 2 weeks (B) and 4 weeks (C) posttransplantation confirmed normal glucose responsiveness in graft-bearing mice (solid line) vs. naïve mice (dashed line) (n = 3). Bar charts on the right panels show area under the IPGTT curves. Error bars indicate SEM.
FIG. 2.
FIG. 2.
Histology of composite islet grafts. Graft-recipient mice were killed at indicated time points posttransplantation. Composite islet grafts were then retrieved and stained with H-E. Untreated (A–C) and mock vector infected (d–F) fibroblast grafts after 1, 2, and 3 weeks posttransplantation, respectively. G–L: IDO-expressing fibroblast grafts after 1–3 and 5–7 weeks posttransplantation. Note that inflammation and cellular infiltration into the graft started in control groups in the second week but, in the IDO group, delayed until the sixth week posttransplantation. Scale bar: 100 μm.
FIG. 3.
FIG. 3.
CD3+ and FOXP3+ cells infiltrating into composite islet grafts and in draining lymph nodes. Graft-recipient mice were killed at indicated time points posttransplantation. Composite islet grafts were then retrieved and subjected to double immunofluorescence staining for CD3 and insulin or FOXP3. A: Composite grafts in untreated, mock vector–infected, and IDO-expressing fibroblast graft at 2 weeks posttransplantation and IDO graft after 5 weeks posttransplantation. The lower panels show high magnification of the indicated area of the upper panels. Note that in the IDO-expressing graft, CD3+ cells accumulated in the border of the graft and kidney tissue but did not infiltrate the graft. Scale bars in the low- and high-magnification panels equal 100 and 20 μm, respectively. B and C: FOXP3 immunofluorescence staining of composite grafts (B) and graft draining lymph nodes (C). Untreated, mock vector, and IDO grafts at 2 weeks posttransplantation and IDO grafts after 5 weeks posttransplantation. Scale bar: 50 μm. (A high-quality digital representation of this figure is available in the online issue.)
FIG. 4.
FIG. 4.
Characterization of graft-infiltrating cells. Graft-recipient mice were killed at indicated time points posttransplantation. Composite islet grafts were then retrieved and processed as described in research design and methods to prepare single-cell suspensions. Phenotype of cells was then assessed using flow cytometry. A: Representative flow cytometry plots comparing CD4+, CD8+, and CD11c+ cells in the grafts. The percent of events in each quadrant of dot plots is indicated. Plots in each row include untreated, mock vector, and IDO grafts at 2 weeks posttransplantation and IDO grafts after 5 weeks posttransplantation. B: frequency of CD3+, CD4+, and CD11c+ cells in grafts. C: Ratio of CD11c+ to CD3+ cells in grafts. Data are means ± SEM. *Significant difference between IDO (week 2) and other groups (P < 0.001; n = 5).
FIG. 5.
FIG. 5.
Cytokine/chemokine expression profile in composite grafts and donor-specific alloantibody production. A: Graft-recipient mice were killed at indicated time points posttransplantation. Composite islet grafts were then retrieved and total RNAs were extracted and subjected to qPCR for cytokines γ-interferon, IL-2, IL-17, IL-4, and IL-10 and chemokines CXCL9 and CXCL10. mRNA levels at indicted time points were standardized by mRNA levels on day 1 posttransplant for each experimental group. Data are means ± SEM. *Significant difference between IDO (week 2) and other groups (P < 0.001; n = 5). B–D: Donor-specific alloantibody production in graft-recipient mice. Naïve donor strain BALB/c thymocytes (H-2d) were incubated with serum collected from B6 (H-2b) mice that received graft with untreated (B), mock vector–infected (C), or IDO-expressing (D) fibroblasts on indicated time points after transplantation (week 1–3 for controls and week 1–7 for IDO group). Binding of alloantibody was assessed by flow cytometry analysis after incubation of FITC-conjugated goat anti-mouse IgG1 or anti-mouse IgG2c antibody. The average percentage of donor cells binding to serum antibodies from three individual recipients per group is expressed.
FIG. 6.
FIG. 6.
FOXP3+ cells in draining lymph nodes and mixed lymphocyte reactions. A: Frequency of FOXP3+ cells in graft draining lymph nodes were calculated by counting FOXP3 immunostained cells (red cells in Fig. 3C) in 10 high-power fields. Data means ± SEM. *Significant difference between IDO (week 2) and other groups (P < 0.001; n = 10). B and C: Graft-draining (renal) lymph node cells from graft recipient (B6; H-2b) mice were harvested and cultured with irradiated islet donor (BALB/c; H-2d) mice (B) and third-party (C3H; H-2k) (C) mice splenocytes at indicated ratios for 96 h. Wells were pulsed with [3H]-thymidine for the last 18 h, and then [3H]-thymidine incorporation to DNA was measured in triplicate. *Significant difference between IDO (week 2) and other groups (P < 0.001; n = 10).
FIG. 7.
FIG. 7.
Stability analysis of IDO transgene expression in composite islet grafts. IDO graft-recipient mice were killed at indicated time points posttransplantation. Composite islet grafts were then retrieved and subjected to immunofluorescence staining or quantitative PCR for IDO. A and B: Immunofluorescence staining of IDO protein (red) in IDO vector–infected grafts after 2 weeks (A) and 5 weeks (B) posttransplantation. Lower panels: high magnification of the indicated area of the upper panels. Scale bars in the low- and high-magnification panels equal 100 and 20 μm, respectively. C: IDO transgene mRNA levels measured by quantitative PCR in IDO vector–infected grafts on days 1–49 posttransplantation. The level of IDO mRNA at each time point was normalized as the percentage of IDO mRNA level on day 1 posttransplantation. *Statistically significant difference compared with IDO mRNA level on day 1 posttransplantation (n = 3; P < 0.001). Error bars indicate SEM. (A high-quality digital representation of this figure is available in the online issue.)

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