AJP - Heart Watch the video to see how APS reaches out to developing nations.
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Heart Circ Physiol 293: H3415-H3423, 2007. First published October 12, 2007; doi:10.1152/ajpheart.00532.2007
0363-6135/07 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
293/6/H3415    most recent
00532.2007v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via Web of Science (5)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Li, J.
Right arrow Articles by Vassalli, G.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Li, J.
Right arrow Articles by Vassalli, G.

Indoleamine 2,3-dioxygenase gene transfer prolongs cardiac allograft survival

Jianping Li,1,* Andrea Meinhardt,1,2,* Marc-Estienne Roehrich,1,2 Dela Golshayan,3,5 Jean Dudler,4 Maria Pagnotta,4 Massimo Trucco,6 and Giuseppe Vassalli1,5

Departments of 1Cardiology, 2Microbiology, 3Nephrology, and 4Rheumatology and 5Transplantation Center, Centre Hospitalier Universitaire Vaudois and Université de Lausanne, Faculté de Biologie et Médicine, Lausanne, Switzerland; and 6Division of Immunogenetics, Department of Pediatrics, Children's Hospital of Pittsburgh, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania

Submitted 25 September 2007 ; accepted in final form 9 October 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Cells that express indoleamine 2,3-dioxygenase (IDO), the rate-limiting enzyme in the catabolism of tryptophan, suppress T cell responses and promote immunological tolerance. However, their role in solid organ transplantation is incompletely understood. We analyzed T cell responses to allogeneic dendritic cells (DCs) genetically modified to express the gene encoding IDO in vitro and IDO gene transfer into the donor heart in a cardiac transplant model in vivo. Bone marrow-derived DCs transduced with the gene encoding IDO produced active IDO protein. This was associated with decreased stimulation of allogeneic T cell proliferation in the mixed leukocyte reaction in vitro. In a cardiac transplant model, adenovirus-mediated IDO gene transfer into the donor heart resulted in transgene expression predominantly in cardiomyocytes. Fischer-344 rat donor hearts transduced with the gene encoding IDO survived for longer periods of time when placed in Lewis rat recipients compared with control vector or vehicle alone [median survival times of 17 (range: 12–22) days vs. 10 (range: 8–14) and 9 (range: 8–13) days, respectively, P < 0.0001]. IDO gene transfer combined with low-dose cyclosporin A (CsA) was more effective than CsA alone (P < 0.05). Numbers of monocytes/macrophages, CD4+ cells, and CD8{alpha}+ cells infiltrating the graft as well as intragraft cytokine transcript levels for IFN-{gamma}, IL-1, TNF-{alpha}, regulated upon secretion, normal T cell expressed, and secreted/chemokine (C-C motif) ligand 5 were decreased after IDO gene transfer (P < 0.05). In conclusion, DCs genetically engineered to overexpress IDO modulate T cell alloresponses in vitro. IDO gene transfer into the donor heart attenuates acute cardiac allograft rejection. Regulation of tryptophan catabolism by means of IDO overexpression may be a useful approach in heart transplantation.

dendritic cells; transforming growth factor-β; regulated on activation, normal T-cell expressed and secreted/C-C chemokine ligand 5

INDOLEAMINE 2,3-DIOXYGENASE (IDO) is a cytosolic heme protein that catalyzes the first and rate-limiting step in the major pathway of catabolism (i.e., the kynurenine pathway) of the essential amino acid tryptophan, the least abundant of the 20 amino acids ({approx}1% of all amino acids). IDO is expressed in many tissues (29, 32), especially in lymphoid organs and the placenta. In humans, few IDO-positive cells are detectable in normal lymphoid tissues, but focal infiltrates are present in tonsils that display features of chronic inflammation (20, 24). IDO expression and activity in peripheral blood-derived monocytes/macrophages and placental trophoblasts are induced by multiple stimuli such as IFN-{gamma}, IL-1, CD40, lipopolysaccharides, and superoxide anions (13, 29). Specific subsets of human and mouse monocyte-derived macrophages and dendritic cells (DCs) that produce IDO in response to IFN-{gamma} are potent suppressors of T cell proliferation (13, 15, 21). In addition, DCs can increase their production of IDO upon cross-linking of CD80/86 on their surfaces with cytotoxic T lymphocyte-associated antigen 4 (CTLA4) (25) or soluble CTLA4-Ig fusion protein (12) and upon transduction with viral vectors (28). In vitro studies have demonstrated that even small numbers of DCs that express IDO endogenously can block large numbers of antigen-activated CD4+ T cells (18, 20). T cells activated via ligation of the T cell receptor (TCR) in tryptophan-free medium are cell cycle arrested at the mid-G1 phase and sensitized to apoptosis when exposed to anti-Fas antibody (15). In addition, tryptophan downstream metabolites induced by IDO directly inhibit CD4+ T cells, CD8+ T cells, and natural killer (NK) cells in vitro (6, 9, 30).

A seminal report by Munn et al. (26) showed that IDO is expressed at the maternal-fetal interface during pregnancy and that specific inhibition of IDO resulted in the rejection of mouse fetuses. IDO appeared to confer immune privileges upon semiallogeneic fetuses through local tryptophan depletion and specific inhibition of antigen-activated T cells. Major roles for IDO in physiological immunoregulation have also been reported in antitumor immunity (23), chronic infection (32), autoimmune responses (8), and cell transplantation (1). However, little is known about the role of IDO in solid organ transplantation. We analyzed IDO gene transfer into DCs and the resulting effect on allogeneic T cell proliferation in the mixed leukocyte reaction (MLR) in vitro. IDO gene transfer into the donor heart was studied in a cardiac transplant model.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Recombinant adenoviruses. The adenoviral vector Ad-IDO was constructed as previously described (1). Briefly, total RNA was isolated from macrophages and was reverse transcription-treated for cDNA synthesis. The murine IDO gene was cloned by PCR using specific primers chosen to include the native ATG transcription start signal as well as the transcription termination signal of the IDO gene and yielded a fragment size of 1.3 kb. Additional nucleotides were included in the primer sequences to create the PmeI restriction endonuclease sites necessary for ligation into the transfer vector used for adenovirus production. After PCR, the IDO product was cloned in a pBluescript vector (Stratagene, La Jolla, CA) using the Prime PCR Cloner Cloning System (5Prime33Prime, Boulder, CO) and inserted into the pQBI-AdCMV5-BFP transfer vector (ADENO-QUEST Kit, Q-Biogene, Carlsbad, CA), which contains the strong cytomegalovirus (CMV)5 promoter driving the expression of the inserted sequence. In addition, this transfer vector contains the coding region for blue fluorescent protein (BFP) under the control of the weaker CMV promoter. Thus, the final transfer vector contains both the IDO gene and the fluorescent marker BFP. Adenoviral vectors containing no transgene (Ad-Null) or CMV promoter-driven β-galactosidase (Ad-LacZ) or an enhanced green fluorescent protein cassette (Ad-EGFP) were used as control and reporter vectors, respectively. Concentrated viral stocks were prepared using two CsCl2 ultracentrifugation gradients and stored in storage buffer [10 mmol/l Tris·HCl (pH 7.4), 1 mmol/l MgCl2, and 10% sucrose]. Titers of Ad-IDO and Ad-Null stock solutions ranged from 8 x 1010 to 3 x 1011 plaque-forming units (PFU)/ml. As a control infusate, virus storage buffer was diluted in PBS in the same proportions as was done for the virus stock.

Transduction of mouse DCs in vitro. Bone marrow (BM) cells were isolated from femurs and tibias of C57Bl/6 (B6) and C3H mice. DCs were generated in IMDM culture medium supplemented with 10% FCS (Euroclone, Pero, Italy), penicillin-streptomycin (Sigma, St. Louis, MO), 50 µM β-mercaptoethanol (AppliChem, Cheshire, CT), and granulocyte-monocyte colony-stimulating factor (GM-CSF; 1% from F558 cell culture supernatant). On day 2, 2 mM aspirin (Hänseler, Herisau, Switzerland) was added to the culture. On day 3, the culture medium was changed and replaced with fresh medium containing aspirin. On day 5, CD11c+ DCs were purified with immunomagnetic microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany) and incubated with adenoviral vectors [multiplicity of infection (MOI) = 100–1,000] in the presence of Polybrene (1.5 µg/ml, Chemicon, Billerica, MA) using a spinoculation technique (1,500 g at 22°C for 2 h). The maturation status of DCs was analyzed by CD80, CD86, and major histocompatibility class II (MHC-II) surface molecule expression analysis by flow cytometry using fluorochrome-conjugated monoclonal antibodies (eBioscience, San Diego, CA).

Western blot analysis. IDO protein expression was assessed by Western blot analysis of cell and tissue extracts using mouse anti-IDO monoclonal antibody (Upstate, Billerica, MA) followed by a horseradish peroxidase-conjugated anti-mouse IgG antibody (Dako, Glostrup, Denmark).

IDO activity assay. IDO catabolic activity was measured as the release of the downstream tryptophan metabolite kynurenine. HEK-293 cells, B6 DCs, and C3H DCs were preincubated with Ad-IDO or Ad-Null at the indicated MOIs or with vehicle alone. After 48 h, cells were apportioned into a 96-well format at 6 x 104 cells/well. After attachment, cells were washed once, and new phenol red-free medium containing 100 µg/ml tryptophan was added. The reaction was stopped after 24 h by the addition of 10 µl of 30% (wt/vol) trichloroacetic acid to 160 µl of cell supernatant, followed by an incubation at 50°C for 30 min. After centrifugation, 100 µl of supernatant were mixed with 100 µl of freshly prepared Ehrlich's solution, and absorbance was read at 492 nm.

In vitro MLR. BM-derived B6 DCs were preincubated with Ad-IDO or Ad-Null at the indicated MOIs or with vehicle alone. After 48 h, DCs were irradiated with 40 Gy and seeded in triplicates at varying densities (5 x 103 or 104 cells/well) in 96-well round-bottomed plates (Corning, Corning, NY). T cells were prepared from spleens of naïve B6 and C3H mice. Splenocytes were washed once in PBS and resuspended in 3 ml ACK buffer (5 min, room temperature); T cells were purified with immunomagnetic microbeads (Miltenyi Biotec). T cells (5 x 104 or 105) were added to DC cultures (final volume: 200 µl/well). On day 3, [3H]thymidine (1 µCi/well, Amersham Pharmacia, Baie d'Urfé, QC, Canada) was added, and cell proliferation was assessed as [3H]thymidine incorporation at 20 h by liquid scintillation counting.

Gene transfer into donor hearts and transplantation. Animal experimentation conformed to the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals (1996) and the Swiss law on animal protection and was approved by the Veterinarian Office of Canton Vaud (Lausanne, Switzerland). Male Fischer-344 (F344) and Lewis (LEW) rats (8–12 wk old, Charles River, L'Arbresle, France) were used as heart donors and recipients, respectively. Ex vivo gene transfer was performed by an intracoronary infusion of vector-containing solution into donor hearts immediately before transplantation. First, hearts were instilled slowly with 500 µl PBS (without Ca2+/Mg2+), followed by 200 µl of virus-containing solution, while allowing efflux through the right pulmonary artery. This artery was then clamped, and an additional 400 µl of virus-containing solution were instilled slowly into the coronary arteries. Vascularized cardiac grafts were placed in the abdominal position. In a preliminary series of experiments, cardiac allografts (n = 4 rats/group) received varying doses of Ad-IDO (109, 1010, and 3 x 1010 PFU), Ad-Null (1010 PFU), or vehicle alone. Three LEW isografts were included as controls. In a second series of experiments, donor hearts (n = 9 rats/group) received Ad-IDO or Ad-Null (1010 PFU each) or vehicle alone. In a third series experiments, recipients (n = 7 rats/group) received low-dose cyclosporin A (CsA; 1.5 mg·kg–1·day–1, Novartis, Basel, Switzerland) on posttransplant days 0–4 alone or in conjunction with IDO gene transfer (1010 PFU). A separate small group of donor hearts (n = 4) received an intramyocardial Ad-IDO administration (1010 PFU in 200 µl virus dilution buffer given at 5 injection sites). Graft survival was monitored by daily abdominal palpation. Rejection (defined as total cessation of heart beating) was confirmed by direct graft examination.

Immunohistochemistry. Gene transfer in donor hearts was visualized by immunostaining of BFP produced by Ad-IDO (1) (this approach was chosen because of suboptimal results using anti-IDO monoclonal antibody). No BFP fluorescence was observed on graft sections by epifluorescence microscopy (in contrast to HEK-293 cells transduced with Ad-IDO in vitro; data not shown). For immunostaining, cryosections were fixed in acetone (–20°C) for 3 min, blocked with PBS, 0.3% Triton X-100, and 1% BSA for 1 h, and incubated with a mixture of goat anti-GFP antibody (which also recognizes BFP due to large sequence homologies between GFP and BFP; 1:700) and rabbit anti-rat von Willebrand factor (vWF) antibody (ab6994, Abcam, Cambridge, UK; 1:1,000) in PBS, 0.3% Triton X-100, and 1% BSA at 4°C overnight. After being washed 3 times with PBS, sections were incubated with Alexa 594-conjugated donkey anti-goat secondary antibody (Molecular Probes, Eugene, OR; 1:400) for 40 min at room temperature, followed by three washes with PBS and an incubation with Alexa 488-conjugated goat anti-rabbit secondary antibody (Molecular Probes; 1:400) for 40 min at room temperature. After being washed three times with PBS, sections were stained with 4',6-diamidino-2-phenylindole (Molecular Probes) for 5 min at room temperature and mounted with Mowiol 4–88 antifade medium (PolySciences, Warrington, PA). Intragraft leukocyte infiltrates were analyzed at day 6 posttransplant. Three series of 8-µm sections per heart were cut at 500-µm steps parallel to the atrioventricular groove from the cardiac apex to the base and stained with the following primary monoclonal antibodies: anti-ED1-like (1C7) detecting monocyte/macrophages, anti-TCR-{alpha}β (R73), anti-CD8{alpha} (OX-8, all from PharMingen, San Diego, CA), anti-CD4 (W3/25, Accurate Chemicals, Westbury, NY), or irrelevant mouse (MOPC-31C; PharMingen), followed by biotin-conjugated rabbit F(ab')2 anti-mouse Ig (Jackson ImmunoResearch, West Grove, PA), StreptABComplex/HRP (Dako), and DAB/H2O2 (Dako). Morphometric analysis was performed in eight microscopic fields (x10) per section using the NIH Image 1.62 program, as previously described (7, 10). Positive-staining areas for each antibody were expressed as percentages of the corresponding myocardial areas of interest.

Real-time RT-PCR. Hearts were excised at day 6 posttransplant, and ~300 mg of tissue were placed in ice-cold PBS, cut into thin pieces, and submerged into RNAlater Stabilization Reagent. Total RNA was extracted with the Qiagen RNeasy kit (Qiagen, Valencia, CA). DNase-treated RNA was used to generate cDNA with reverse transcriptase Omniscript (Qiagen), random hexamer (Promega, Madison, WI), and RNase inhibitor (Roche, Basel, Switzerland). cDNA equivalent to 100 ng total RNA was used for each PCR. Cytokine transcript levels were measured by real-time PCR (Rotor-gene multifilter real-time cycler, Corbett Research, Sydney, Australia) using SYBR green dye and hypoxanthine phosphoribosyltransferase (HPRT) as a reference gene. Primer sequences were as follows: IFN-{gamma}, 5'-TCATGGCCCTCTCTGGCTGTTAC-3' (sense) and 5'-CAAGAGGAGGCTCTTTCCTTCCATAG-3' (antisense); IL-1β, 5'-CTTCAAATCTCACAGCAGCATCTCG-3' (sense) and 5'-TCCACGGGCAAGACATAGGTAGC-3' (antisense); TNF-{alpha}, 5'-CTGTGCCTCAGCCTCTTCTCATTC-3' (sense) and 5'-TTGGGAACTTCTCCTCCTTGTTGG-3' (antisense); transforming growth factor (TGF)-β, 5'-CTAATGGTGGACCGCAACAACG-3' (sense) and 5'-TCTGGCACTGCTTCCCGAATG-3' (antisense); regulated on activation, normal T cell expressed and secreted/C-C chemokine ligand 5 (RANTES/CCL5), 5'-CCATATGGCTGCGACACCACTC-3' (sense) and 5'-GCACACACTTGGCGGTTCCTT-3' (antisense); and HPRT, 5'-CTCATGGACTGATTATGGACAGGACTG-3' (sense) and 5'-CAGCGCTTTAATGTAATCCAGCAGGTC-3' (antisense). Results were expressed as cytokine-to-HPRT mRNA copy ratios.

Statistical analysis. Graft survival data are shown as median survival times (MST) and ranges and as Kaplan-Meier cumulative survival curves. The statistical significance of differences in graft survival between groups was evaluated using the Mantel-Cox log rank test. The statistical significance of differences in intragraft leukocyte infiltrates and cytokine mRNA expression between Ad-IDO and Ad-Null was evaluated using the Mann-Whitney U-test.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
DCs transduced with the gene encoding IDO inhibit T cell proliferation in vitro. The purity of BM-derived DCs, as assessed by flow cytometry analysis of CD11c expression, was {approx}90%. The surface molecule profile of DCs before viral transduction was CD86lowCD80+MHC-IIlow (data not shown), reflecting a partially immature phenotype. Gene transfer efficiency, as assessed by flow cytometry analysis of EGFP expression in DCs preincubated with Ad-EGFP, was {approx}70% (Fig. 1A). Adenovirus induced DC maturation, as evidenced by CD86 upregulation (Fig. 1A), which was partially prevented by aspirin. IDO protein expression was detected by Western blot analysis in DCs preincubated with Ad-IDO. Lower levels of endogenous IDO protein were detectable in DCs preincubated with Ad-Null (Fig. 1B), in line with previous data showing IDO upregulation in maturing DCs (5). Supernatants of HEK-293 cells and DCs preincubated with Ad-IDO showed increased L-kynurenine levels, reflecting IDO activity (Fig. 1C). In the in vitro MLR, IDO-overexpressing DCs induced weaker allospecific T cell proliferation compared with DCs preincubated with Ad-Null (Fig. 1D).


Figure 1
View larger version (21K):
[in this window]
[in a new window]

  		Fig. 1. A: adenoviral transduction of bone marrow (BM)-derived mouse dendritic cells (DCs). Flow cytometry analysis of DCs for green fluorescence protein (GFP) and CD86 expression in DCs preincubated with adenovirus (Ad) containing an enhanced GFP (EGFP) cassette or no virus in the presence or absence of aspirine (ASA). B: detection of murine indoleamine 2,3-dioxygenase (IDO) protein expression in vitro. Western blots of protein extracts from HEK-293 cells and BM-derived mouse DCs were preincubated with Ad-IDO or Ad-Null at the indicated multiplicities of infection (MOIs). IDO is a 43-kDa protein. C: measurements of IDO activity in vitro. Spectrophotometric analysis of kynurenine production by HEK-293 cells as well as C57Bl/6 (B6) and C3H DCs preincubated with Ad-IDO, Ad-Null, or vehicle alone are shown (note the different graph scales for the two cell types). D: allospecific T cell proliferation in the in vitro mixed leukocyte reaction [in counts/min (cpm)]. B6 DCs (5 x 103 or 104) preincubated with Ad-IDO, Ad-Null, or vehicle alone were cocultured with splenic T cells from B6 or C3H mice. Representative data from 1 of 3 experiments are shown.

 
IDO gene transfer prolongs cardiac allograft survival in vivo. IDO protein was detectable by Western blot analysis in graft tissue extracts at 2 and 10 days after IDO gene transfer, with an approximately sixfold decrease between the two time points (Fig. 2A). No IDO protein was detectable in nontransplanted hearts and cardiac allografts that received Ad-Null. Cardiomyocytes accounted for almost all of the BFP-positive graft cells after IDO gene transfer (Fig. 3), which were undetectable in control hearts. Virtually no BFP-positive endothelial cells were observed by double labeling with anti-vWF antibody. In preliminary experiments in a small number of animals (n = 4 rats/group), Ad-IDO at the 1010 PFU dose prolonged allograft survival to a slightly larger extent than the 109 PFU dose, whereas a further increase in the virus dose to 3 x 1010 PFU did not result in further improvement of graft survival (Table 1). Therefore, the 1010 PFU dose was used in the second series of experiments involving a larger number of animals (n = 9 rats/group). Ad-IDO prolonged graft survival compared with Ad-Null and vehicle alone [MST of 17 (range: 12–22) days vs. 10 (range: 8–14) and 9 (range: 8–13) days, respectively, P < 0.0001; Fig. 2B]. In the third series of experiments (n = 7 rats/group), Ad-IDO (1010 PFU) in conjuction with low-dose CsA prolonged graft survival to a larger extent than CsA alone [MST of 19 (range: 16–24) days vs. 15 (range: 13–17) days, P < 0.05; Fig. 2C]. In a separate group, an intramyocardial injection of Ad-IDO into the donor heart showed similar effects on graft survival compared with the intracoronary route of administration [MST of 17 (range: 15–18) days].


Figure 2
View larger version (60K):
[in this window]
[in a new window]

  		Fig. 2. Immunohistological analysis of gene transfer in cardiac allografts. A–D: control vector (magnification: x20). E–H: Ad-IDO (magnification: x20). I–L: Ad-IDO (magnification: x40). A, E, and I: immunostaining for blue fluorescent protein (red) expressed from Ad-IDO. B, F, and J: immunostaining for von Willebrand factor (green) expressed on endothelial cells (a small vessel is visible in B). C, G, and K: merge images. D and H: nuclear staining with 4',6-diamidino-2-phenylindole (DAPI). L: negative control. Transduced cells are striated fibers (i.e., cardiomyocytes morphology) but not endothelial cells.

 

Figure 3
View larger version (17K):
[in this window]
[in a new window]

  		Fig. 3. IDO expression in cardiac allografts and graft survival. A: Western blot of protein extracts from HEK-293 cells preincubated with Ad-IDO as a positive control (showing a faint 43-kDa band) and from cardiac allografts receiving Ad-null, no virus, or Ad-IDO (2 and 10 days after gene transfer). B: Kaplan-Meier survival plots for event times for Ad-IDO, Ad-Null, and vehicle alone (P < 0.0001 for Ad-IDO vs. both control goups). C: IDO gene transfer combined with low-dose cyclosporin A (CsA) vs. CsA alone (P < 0.05; same data for control groups as in B).

 

View this table:
[in this window]
[in a new window]

  		Table 1. Allograft survival after the first series of heart transplant experiments

 
IDO gene transfer attenuates graft infiltration by leukocytes. IDO gene transfer was associated with decreased graft infiltration by ED1-like+ monocytes/macrophages (4.7-fold), CD4+ cells (5.1-fold), TCR-{alpha}β+ cells (3.0-fold), and CD8{alpha}+ cells (3.0-fold) compared with control vector (P < 0.05 for each cell marker; Fig. 4).


Figure 4
View larger version (106K):
[in this window]
[in a new window]

  		Fig. 4. Mononuclear cell infiltrates in cardiac allografts. Top: immunostaining for CD4+ cells, ED1-like+ monocytes/macrophages, CD8{alpha}+ cells, and T cell receptor (TCR)-{alpha}β-bearing T cells. The high-magnification images are shown for better visualization of positive-staining cells. Bottom: quantitative analysis of inflammatory cell infiltrates staining positive for the same surface markers. Data are means ± SD of percent myocardial areas.

 
IDO gene transfer inhibits intragraft cytokine expression. IDO gene transfer was associated with decreased intragraft transcripts levels for IFN-{gamma} (3-fold), IL-1β (6-fold), RANTES/CCL5 (7-fold), TNF-{alpha} (8-fold), and TGF-β (14-fold) compared with control vector (P < 0.05 for each cytokine; Fig. 5).


Figure 5
View larger version (13K):
[in this window]
[in a new window]

  		Fig. 5. Real-time RT-PCR analysis of intragraft cytokine expression. Data are cytokine mRNA copy numbers normalized for hypoxanthine phosphoribosyltransferase mRNA copy numbers (logarithmic graph scale). Fold decreases in the Ad-IDO vs. Ad-Null group are indicated. TGF-β, transforming growth factor-β; RANTES, regulated upon secretion, normal T cell expressed, and secreted.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
IDO modulates various immune responses, including antiself, antifetal, and antitumor responses. We evaluated DCs genetically modified with the gene encoding IDO in vitro as well as IDO gene transfer into the donor heart in a cardiac transplant model.

Previous studies have reported that specific subsets of DCs and monocyte-derived macrophages that produce IDO in response to IFN-{gamma} are potent suppressors of T cell proliferation (13, 15, 21, 30). We analyzed the impact of transgenic IDO overexpression by BM-derived murine DCs on allospecific T cell stimulation in vitro. DCs transduced with the gene encoding IDO produced functional IDO, which attenuated allospecific T cell proliferation in the MLR in vitro. However, in vitro cell manipulation and adenoviral transduction resulted in DC maturation, which counterbalanced the inhibitory effect of IDO on T cell responses.

In a cardiac transplant model, IDO ex vivo gene delivery to the donor heart reduced graft infiltration by inflammatory cells as well as intragraft cytokine expression, resulting in a moderate yet significant prolongation of allograft survival. IDO gene transfer in conjunction with a short course of low-dose CsA was more effective than CsA alone. Immunohistologically, cardiomyocytes accounted for almost all of the gene-transduced cells in the graft, suggesting that this may be the cell type responsible for the protective effect. As mentioned above, initial reports focused on IFN-{gamma}-mediated induction of IDO-positive subsets of regulatory DCs and monocyte-derived macrophages as well as on IDO-producing placental trophoblasts. However, recent evidence suggests that endogenous or transgenic IDO expression in a variety of cell types may suppress T cell responses via tryptophan catabolism. For instance, BM stromal cells have been shown to suppress allogeneic T cell responses via IDO-mediated tryptophan degradation (17). Tumor cells transfected with the gene encoding IDO inhibited T cell proliferation in the microenvironment (19). Islet cells transduced with the gene encoding IDO induced tryptophan depletion from culture medium, suppressed T cell proliferation in vitro, and survived for extended periods of time after transplantation into nonobese diabetic (NOD)/severe combined immunodeficient (SCID) recipients and adoptive transfer of NOD diabetogenic T cells (1). In a recent study, human vascular endothelial cells transfected with the gene encoding IDO were incapable of stimulating allogeneic T cell responses and induced anergy of allospecific T cells in vitro (4). Murine corneal endothelial cells transduced with the gene encoding IDO expressed functional IDO, which inhibited allogeneic T cell proliferation and prolonged corneal graft survival (3). Finally, lung cells transfected with the gene encoding IDO inhibited inflammation-related oxidative stress and TGF-β-induced fibrosis in vitro as well as lung allograft fibrosis in a rat model (16). Conversely, specific IDO inhibition accelerated skin (27), but not corneal (3), allograft rejection in mice. Collectively, these data are consistent with transgenic IDO expression in multiple different cell types suppressing local T cell responses. Our data suggest that this may also be the case for myocytes in cardiac grafts. Two mechanisms have been evoked to explain these findings: namely, tryptophan starvation and tryptophan downstream metabolites induced by IDO. The first one is based on the observation that T cells activated via TCR ligation in tryptophan-free medium are cell cycle arrested at the mid-G1 stage and sensitized to apoptosis when exposed to anti-Fas antibody (15) and that tryptophan supplementation reverses IDO-mediated T cell suppression (22). A GCN2 stress-response kinase mediates proliferative arrest and anergy in T cells in response to IDO (22), but exactly how IDO creates the stress that activates GCN2 in T cells is not known. The second mechanism involves tryptophan downstream metabolites induced by IDO, which directly inhibit CD4+ cells, CD8+ cells, and NK cells in vitro (6, 9, 30). Both tryptophan depletion and downstream tryptophan metabolites induce TCR {zeta}-chain downregulation and a regulatory phenotype in naïve T cells (9).

In our model, there is a remote possibility that rare DCs in the donor heart were transduced with the gene encoding IDO, left the graft, migrated into the host spleen (14), and inhibited allospecific T cell responses. However, this mechanism seems unlikely for several reasons. Our in vitro data indicate that efficient gene transduction into DCs requires high viral MOIs that exceed by far those achievable in the donor heart. In addition, we have shown that adenovirus-induced DC maturation counterbalances the inhibitory effect of IDO overexpression in the MLR in vitro. It is therefore unlikely that gene-transduced DCs contributed to the beneficial effect of IDO gene transfer in vitro.

In our model, IDO gene transfer conferred only moderate protection upon cardiac allografts. This may be due, in part, to methodological aspects potentially amenable to a solution in the future. These aspects include vector tropism, gene transfer efficiency, vector toxicity, and delayed onset, and short duration of IDO transgene expression. Consistent with the low tropism of adenovirus vectors for the endothelium, we did not observe significant gene transfer into graft endothelial cells in our model. In this regard, whereas the vascular endothelium expresses little endogenous IDO, transgenic IDO expression by endothelial cells locally suppresses T cell responses (4). Furthermore, adenovirus vectors mediate tissue inflammation, which may offset the beneficial effects of IDO overexpression. The kinetics of transgene expression and tryptophan degradation by IDO also need to be considered. The production of IDO protein in gene-transduced cells starts approximately hours after gene delivery and presumably reaches peak levels one to several days thereafter. In addition, several hours to a few days are needed for IDO-mediated tryptophan degradation (Fig. 1C). The immunomodulatory effect presumably depends on the degree of tryptophan depletion and/or tryptophan metabolite production but cannot be achieved in the first few hours after transplantation. Therefore, a potential role for IDO gene therapy as an adjunctive treatment is conceivable, but the use of immunosuppressive agents would remain mandatory, at least in the early posttransplant period. Finally, adenoviral transgene expression is short lived (Fig. 3A). Other vectors, such as adeno-associated virus (AAV) vectors, have a potential for sustained transgene expression [we have observed AAV-mediated EGFP expression during 1 yr in mouse hearts (31)].

Clearly, several methodological aspects of IDO gene therapy need to be refined to optimize the therapeutic effect. The present proof of principle study suggests that the regulation of tryptophan metabolism by IDO gene transfer may be a useful approach in heart transplantation.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
Financial support was obtained from the Swiss National Science Foundation, the Teo Rossi di Montelera Foundation, the Swiss Cardiology Foundation, the Novartis Research Foundation, and the Lausanne Transplant Foundation.


   ACKNOWLEDGMENTS
 
We thank Dr. Eleonora Simeoni, Dr. Sylvain Fleury, and Dr. Emma Fiorini for scientific help and Kaethy Mujynya-Ludunge for technical help.

Present address of J. Li: Transplant Research Laboratories, Novartis, Basel, Switzerland.


   FOOTNOTES
 

Address for reprint requests and other correspondence: G. Vassalli, Centre Hospitalier Universitaire Vaudois, BH10, Lausanne 1011, Switzerland (e-mail: giuseppe.vassalli{at}chuv.chuv.ch)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

* J. Li and A. Meinhardt contributed equally to this work. Back


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Alexander AM, Crawford M, Bertera S, Rudert WA, Takikawa O, Robbins PD, Trucco M. Indoleamine 2,3-dioxygenase expression in transplanted NOD islets prolongs graft survival after adoptive transfer of diabetogenic splenocytes. Diabetes 51: 356–365, 2002.[Abstract/Free Full Text]
  2. Azzawi M, Hasleton PS, Geraghty PJ, Yonan N, Krysiak P, El-Gammal A, Deiraniya AK, Hutchinson IV. RANTES chemokine expression is related to acute cardiac cellular rejection and infiltration by CD45RO T-lymphocytes and macrophages. J Heart Lung Transplant 17: 881–887, 1998.[Web of Science][Medline]
  3. Beutelspacher SC, Pillai R, Watson MP, Tan PH, Tsang J, McClure MO, George AJ, Larkin DF. Function of indoleamine 2,3-dioxygenase in corneal allograft rejection and prolongation of allograft survival by over-expression. Eur J Immunol 36: 690–700, 2006.[CrossRef][Web of Science][Medline]
  4. Beutelspacher SC, Tan PH, McClure MO, Larkin DFP, Lechler RI, George AJT. Expression of indoleamine 2,3-dioxygenase (IDO) by endothelial cells: implications for the control of alloresponses. Am J Transplant 6: 1320–1330, 2006.[CrossRef][Web of Science][Medline]
  5. Braun D, Longman RS, Albert ML. A two-step induction of indoleamine 2,3 dioxygenase (IDO) activity during dendritic-cell maturation. Blood 106: 2375–2381, 2005.[Abstract/Free Full Text]
  6. Della Chiesa M, Carlomagno S, Frumento G, Balsamo M, Cantoni C, Conte R, Moretta L, Moretta A, Vitale M. The tryptophan catabolite L-kynurenine inhibits the surface expression of NKp46- and NKG2D-activating receptors and regulates NK-cell function. Blood 108: 4118–4125, 2006.[Abstract/Free Full Text]
  7. Dudler J, Li J, Pagnotta M, Pascual M, von Segesser LK, Vassalli G. Gene transfer of programmed death ligand-1. Ig prolongs cardiac allograft survival. Transplantation 82: 1733–1737, 2006.[CrossRef][Web of Science][Medline]
  8. Fallarino F, Grohmann U, You S, McGrath BC, Cavener DR, Vacca C, Orabona C, Bianchi R, Belladonna ML, Volpi C, Fioretti MC, Puccetti P. Tryptophan catabolism generates autoimmune-preventive regulatory T cells. Transpl Immunol 17: 58–60, 2006.[CrossRef][Web of Science][Medline]
  9. Fallarino F, Grohmann U, You S, McGrath BC, Cavener DR, Vacca C, Orabona C, Bianchi R, Belladonna ML, Volpi C, Santamaria P, Fioretti MC, Puccetti P. The combined effects of tryptophan starvation and tryptophan catabolites down-regulate T cell receptor zeta-chain and induce a regulatory phenotype in naive T cells. J Immunol 176: 6752–6761, 2006.[Abstract/Free Full Text]
  10. Fleury S, Li J, Simeoni E, Driscoll R, von Segesser LK, Kappenberger L, Vassalli G. Gene transfer of RANTES and MCP-1 chemokine receptor antagonists prolongs cardiac allograft survival. Gene Ther 13: 1104–1109, 2006.[CrossRef][Web of Science][Medline]
  11. Funeshima N, Fujino M, Kitazawa Y, Hara Y, Hara Y, Hayakawa K, Okuyama T, Kimura H, Li XK. Inhibition of allogeneic T-cell responses by dendritic cells expressing transduced indoleamine 2,3-dioxygenase. J Gene Med 7: 565–575, 2005.[CrossRef][Web of Science][Medline]
  12. Grohmann U, Orabona C, Fallarino F, Vacca C, Calcinaro F, Falorni A, Candeloro P, Belladonna ML, Bianchi R, Fioretti MC, Puccetti P. CTLA-4-Ig regulates tryptophan catabolism in vivo. Nat Immun 2: 1097–1101, 2002.[Web of Science]
  13. Hwu P, Du MX, Lapointe R, Do M, Taylor MW, Young HA. Indoleamine 2,3-dioxygenase production by human dendritic cells results in the inhibition of T cell proliferation. J Immunol 164: 3596–3599, 2000.[Abstract/Free Full Text]
  14. Larsen CP, Morris PJ, Austyn JM. Migration of dendritic leukocytes from cardiac allografts into host spleens. A novel pathway for initiation of rejection. J Exp Med 171: 307–314, 1990.[Abstract/Free Full Text]
  15. Lee GK, Park HJ, MacLeod M, Chandler P, Munn DH, Mellor AL. Tryptophan deprivation sensitizes activated T cells to apoptosis prior to cell division. Immunology 107: 452–460, 2002.[CrossRef][Web of Science][Medline]
  16. Liu H, Liu L, Fletcher BS, Visner GA. Sleeping Beauty-based gene therapy with indoleamine 2,3-dioxygenase inhibits lung allograft fibrosis. FASEB J 20: E1694–E1703, 2006.
  17. Meisel R, Zibert A, Laryea M, Gobel U, Daubener W, Dilloo D. Human bone marrow stromal cells inhibit allogeneic T-cell responses by indoleamine 2,3-dioxygenase-mediated tryptophan degradation. Blood 103: 4619–4621, 2004.[Abstract/Free Full Text]
  18. Mellor AL, Chandler P, Baban B, Hansen AM, Marshall B, Pihkala J, Waldmann H, Cobbold S, Adams E, Munn DH. Specific subsets of murine dendritic cells acquire potent T cell regulatory functions following CTLA4-mediated induction of indoleamine 2,3-dioxygenase. Int Immunol 16: 1391–1401, 2004.[Abstract/Free Full Text]
  19. Mellor AL, Keskin DB, Johnson T, Chandler P, Munn DH. Cells expressing indoleamine 2,3-dioxygenase inhibit T cell responses. J Immunol 168: 3771–3776, 2002.[Abstract/Free Full Text]
  20. Munn DH. Tolerogenic antigen-presenting cells. Ann NY Acad Sci 961: 343–345, 2002.[Web of Science][Medline]
  21. Munn DH, Shafizadeh E, Attwood JT, Bondarev I, Pashine A, Mellor AL. Inhibition of T cell proliferation by macrophage tryptophan catabolism. J Exp Med 189: 1363–1372, 1999.[Abstract/Free Full Text]
  22. Munn DH, Sharma MD, Baban B, Harding HP, Zhang Y, Ron D, Mellor AL. GCN2 kinase in T cells mediates proliferative arrest and anergy induction in response to indoleamine 2,3-dioxygenase. Immunity 22: 633–624, 2005.[CrossRef][Web of Science][Medline]
  23. Munn DH, Sharma MD, Hou D, Baban B, Lee JR, Antonia SJ, Messina JL, Chandler P, Koni PA, Mellor AL. Expression of indoleamine 2,3-dioxygenase by plasmacytoid dendritic cells in tumor-draining lymph nodes. J Clin Invest 114: 280–290, 2004 (Erratum. J Clin Invest 114: 599, 2004).[CrossRef][Web of Science][Medline]
  24. Munn AH, Sharma MD, Lee JR, Jhaver KG, Johnson TS, Keskin DB, Marshall B, Chandler P, Antonia SJ, Burgess R, Slingluff CL Jr, Mellor AL. Potential regulatory function of human dendritic cells expressing indoleamine 2,3-dioxygenase. Science 297: 1867–1870, 2002.[Abstract/Free Full Text]
  25. Munn DH, Sharma MD, Mellor AL. Ligation of B7-1/B7-2 by human CD4+ T cells triggers indoleamine 2,3-dioxygenase activity in dendritic cells. J Immunol 172: 4100–4110, 2004.[Abstract/Free Full Text]
  26. Munn DH, Zhou M, Attwood JT, Bondarev I, Conway SJ, Marshall B, Brown C, Mellor AL. Prevention of allogeneic fetal rejection by tryptophan catabolism. Science 281: 1191–1193, 1998.[Abstract/Free Full Text]
  27. Sakurai K, Zou JP, Torres NI, Tschetter JR, Kim HS, Shearer GM. Study of the effect of expressing indoleamine 2,3-dioxygenase on murine mixed lymphocyte reactions and skin allograft rejection. Transplant Proc 34: 3271–3273, 2002.[CrossRef][Web of Science][Medline]
  28. Tan PH, Beutelspacher SC, Wang YH, McClure MO, Ritter MA, Lombardi G, George AJ. Immunolipoplexes: an efficient, nonviral alternative for transfection of human dendritic cells with potential for clinical vaccination. Mol Ther 11: 790–800, 2005.[CrossRef][Web of Science][Medline]
  29. Taylor MW, Feng G. Relationship between interferon-{gamma}, indoleamine 2,3-dioxygenase, and tryptophan catabolism. FASEB J 5: 2516–2522, 1991.[Abstract]
  30. Terness P, Bauer TM, Rose L, Dufter C, Watzlik A, Simon H, Opelz G. Inhibition of allogeneic T cell proliferation by indoleamine 2,3-dioxygenase-expressing dendritic cells: mediation of suppression by tryptophan metabolites. J Exp Med 196: 447–457, 2002.[Abstract/Free Full Text]
  31. Vassalli G, Büeler H, Dudler J, von Segesser LK, Kappenberger L. Adeno-associated virus (AAV) vectors achieve prolonged transgene expression in mouse myocardium and arteries in vivo: a comparative study with adenovirus vectors. Int J Cardiol 90: 231–240, 2003.
  32. Yoshida R, Nukiwa T, Watanabe Y, Fujiwara M, Hirata F, Hayaishi O. Regulation of indoleamine 2,3-dioxygenase activity in the small intestine and the epididymis of mice. Arch Biochem Biophys 203: 343–351, 1980.[CrossRef][Web of Science][Medline]




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
293/6/H3415    most recent
00532.2007v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via Web of Science (5)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Li, J.
Right arrow Articles by Vassalli, G.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Li, J.
Right arrow Articles by Vassalli, G.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2007 by the American Physiological Society.