IDO and Regulatory T Cell Support Are Critical for Cytotoxic T Lymphocyte-Associated Ag-4 Ig-Mediated Long-Term Solid Organ Allograft Survival

Robert Sucher, Klaus Fischler, Rupert Oberhuber, Irmgard Kronberger, Christian Margreiter, Robert Ollinger, Stefan Schneeberger, Dietmar Fuchs, Ernst R. Werner, Katrin Watschinger, Bettina Zelger, George Tellides, Nina Pilat, Johann Pratschke, Raimund Margreiter, Thomas Wekerle and Gerald Brandacher
J Immunol January 1, 2012, 188 (1) 37-46; DOI: https://doi.org/10.4049/jimmunol.1002777

Abstract

Costimulatory blockade of CD28-B7 interaction with CTLA4Ig is a well-established strategy to induce transplantation tolerance. Although previous in vitro studies suggest that CTLA4Ig upregulates expression of the immunoregulatory enzyme IDO in dendritic cells, the relationship of CTLA4Ig and IDO in in vivo organ transplantation remains unclear. In this study, we studied whether concerted immunomodulation in vivo by CTLA4Ig depends on IDO. C57BL/6 recipients receiving a fully MHC-mismatched BALB/c heart graft treated with CTLA4Ig + donor-specific transfusion showed indefinite graft survival (>100 d) without signs of chronic rejection or donor specific Ab formation. Recipients with long-term surviving grafts had significantly higher systemic IDO activity as compared with rejectors, which markedly correlated with intragraft IDO and Foxp3 levels. IDO inhibition with 1-methyl-dl-tryptophan, either at transplant or at postoperative day 50, abrogated CTLA4Ig + DST-induced long-term graft survival. Importantly, IDO1 knockout recipients experienced acute rejection and graft survival comparable to controls. In addition, αCD25 mAb-mediated depletion of regulatory T cells (Tregs) resulted in decreased IDO activity and again prevented CTLA4Ig + DST induced indefinite graft survival. Our results suggest that CTLA4Ig-induced tolerance to murine cardiac allografts is critically dependent on synergistic cross-linked interplay of IDO and Tregs. These results have important implications for the clinical development of this costimulatory blocker.

Despite recent advances in immunosuppressive drug and protocol development, long-term side effects are still a major problem after solid organ transplantation. Therefore there is a continued search to develop alternative immunosuppressive approaches to more selectively regulate the alloimmune response and hence limit systemic toxicities. However, the ultimate goal remains strategies to induce immune tolerance, defined as donor Ag-specific unresponsiveness without the need for lifelong immunosuppression (1).

In this regard, an increased understanding of the molecular pathways involved in Ag presentation and T cell activation has provided various novel targets for therapeutic intervention in transplantation over the past decade. One of the most promising approaches identified was blockade of T cell costimulatory pathways such as the CD28-B7 pathway. Cytotoxic T lymphocyte-associated Ag-4 Ig (CTLA4Ig) is an engineered fusion protein comprised of the extracellular human or murine domain of CTLA4 and the hinge CH2 and CH3 domains of a human or mouse IgG that binds to B7 molecules with high affinity thus preventing CTLA4-CD28 engagement and leading to partial T cell activation and T cell anergy (2).

Costimulatory blockade by use of CTLA4Ig with or without the concomitant administration of donor cells has been demonstrated as a promising strategy to prevent acute and chronic rejection and to induce tolerance in various small and large animal models (3, 4). Recently a novel mutant of CTLA4Ig, LEA29Y, with increased binding affinity to B7 has been generated and successfully introduced into human trials with favorable results (5).

However, the mechanistic and molecular basis of CTLA4Ig function in solid organ transplantation remains poorly understood. For the particular case of islet cell transplantation, Grohmann et al. (6) proposed that the immunomodulatory enzyme IDO is the mediator of CTLA4Ig-induced tolerance via reverse B7 signaling in dendritic cells (DCs). In addition, Mellor et al. and Baban et al. (7–9) published a series of articles describing the effects of CTLA4Ig administration on IDO induction in a subset of splenic DCs that led to T cell suppression in vivo. IDO is induced in various cell types and tissues by cytokines such as IFN-γ and catalyzes the initial and rate-limiting step in the degradation of the essential amino acid tryptophan (10). Via tryptophan depletion and the production of proapoptotic downstream metabolites, IDO suppresses adaptive T cell-mediated immunity and provides the common basis for tolerance induction in pregnancy, autoimmunity, tumor immunosurveillance, and transplantation (10–13).

Growing evidence also indicates that IDO contributes to the immunoregulatory effects of CD4+CD25+Foxp3+ regulatory T cells (Tregs) (14, 15). Tregs, which constitutively express CTLA4, have been shown to lead to IDO production by DCs following B7 engagement and thereby facilitate their regulatory function. In addition, costimulatory blockade can lead to the generation and emergence of Tregs and thereby induce a state of tolerance (16). However, it is unknown whether those mechanisms are operative in CTLA4Ig-mediated models of transplantation tolerance to solid organ transplants. Therefore the aim of this study was to investigate whether CTLA4Ig-mediated costimulatory blockade in vivo depends on and requires both IDO and Tregs for its immunosuppressive and protolerogenic action.

Materials and Methods

Animals

Male inbred BALB/c and C57BL6 mice weighting 20–25 g were obtained from Harlan Winkelmann (Borchen, Germany) and used for transplant experiments. C57BL6IDO1−/− mice were provided by the Department of Surgery, Yale University School of Medicine (New Haven, CT). hCTLA4Ig (abatacept) was provided by Bristol-Myers-Squibb (Princton, NJ) and given i.p. for all in vivo experiments. All animals received human care in compliance with the Principles of Laboratory Animal Care formulated by the National Society for Medical Research and the Guide for the Care and Use of Laboratory Animals prepared by the National Academy of Sciences and published by the National Institutes of Health (NIH Publication No. 86-23, revised 1985). All experiments were approved by the Austrian Ministry of Education, Science, and Culture (BMWF-66.011/0144-II/10b/2009).

Heterotopic cervical heart transplantation

BALB/c hearts were transplanted heterotopically to C57BL/6 (WT/IDO1−/−) using a modified cuff technique. Graft survival was assessed daily by visual inspection and palpation. Treatment of recipient mice consisted of no treatment, DST (5 × 106 BALB/c splenocytes i.v. on day 0), CTLA4Ig (0.5 mg i.p. on day 0; 0.25 mg on days 2, 4, and 6), CTLA4Ig (0.5 mg i.p. on day 0; 0.25 mg on days 2, 4, and 6) plus DST (5 × 106 splenocytes i.v. on day 0), CTLA4Ig (0.5 mg i.p. on day 0; 0.25 mg on days 2, 4, and 6) plus DST (5 × 106 splenocytes i.v. on day 0) plus 1-MT (200 mg, 9-d release pellets implanted on day 0), CTLA4Ig (0.5 mg i.p. on day 0; 0.25 mg on days 2, 4, and 6) plus DST (5 × 106 splenocytes i.v. on day 0) plus 1-MT (200 mg, 9-d release pellets implanted on day 50), CTLA4Ig (0.5 mg i.p. on day 0; 0.25 mg on days 2, 4, and 6) plus DST (5 × 106 splenocytes i.v. on day 0; IDO−/−), anti-CD25mAb (PC61, 0.5 mg i.p. on day 5) plus CTLA4Ig (0.5 mg i.p. on day 0; 0.25 mg on days 2, 4, and 6) plus DST (5 × 106 splenocytes i.v. on day 0). Day 0 is defined as the day of heart transplantation.

Donor-specific transfusion

Spleen tissue was fragmented in PBS (PAA Laboratories, Linz, Austria) and RBCs were lysed using an ACK lysis buffer. Donor-specific splenocytes were cultured for 24 h in RPMI 1640 supplemented with 10% heat-inactivated FCS, 100 U/ml penicillin, 100 μg/ml streptomycin, 2 mM l-glutamine, 10 mM HEPES, 0.1 mM nonessential amino acids, 1 mM sodium pyruvate, and 5 μg/ml Con A (all from Sigma Aldrich, Vienna, Austria). For DST, 5 × 106 cells were injected i.v. at the time of transplantation.

Tryptophan and kynurenine measurement

Tryptophan and kynurenine concentrations in serum were determined by reversed-phase HPLC on days 0, 5, 10, 15, and 100 or at times of allograft rejection as described earlier (17). Specimens were deproteinized with trichloroacetic acid and were separated on reversed phase C18 material using 0.015 mol/l potassium phosphate buffer (pH 6.4). Tryptophan was monitored by means of its native fluorescence at 285 nm excitation and 360 nm emission wavelengths; kynurenine was detected by ultraviolet-absorption at 365 nm wavelength in the same chromatographic run. Finally, kynurenine/tryptophan (kyn/trp) was calculated as an indirect estimate of IDO activity by dividing kynurenine concentrations (μmol/l) by tryptophan concentrations (mmol/l).

Histology

Tissues were fixed in 10% neutral buffered formalin and embedded in paraffin; 5-μm sections were stained with H&E and scored according to the International Society for Heart and Lung Transplantation (ISHLT) rejection score.

Immunohistochemistry

IDO and Foxp3 immunohistochemistry was performed on 4–6-μm heart sections. Sections were incubated for 30 min with polyclonal anti-indoleamine 2,3 dioxygenase (catalog no. ALX-210-432; Enzo Life Sciences, Farmingdale, NY) and Foxp3 (catalog no. 14-4777-82; Biocare Medical, Concord, CA) Ab. Immunostaining of all mouse tissue specimens was performed using an automated method on the Ventana using the iVIEW DAB indirect biotin streptavidin detection system (Ventana Medical Systems, Tucson, AZ). Immunoreactivity was semiquantitatively scored in a blinded fashion as follows: 0, no immunostaining in any vessels or cells; 1, slight immunostaining in few cells or vessels; 2, slight immunostaining in many cells or vessels or strong staining in a few cells or vessels; 3, strong immunostaining in many cells or vessels (18).

Quantitative RT-PCR

For real-time quantitative PCR (qPCR), 500 ng of total RNA isolated by aid of the Trizol reagent (Invitrogen) was reverse-transcribed using M-MLV RNAse H-, (Promega), and gene expression quantified in relation to 18S rRNA using the Taqman-technique in an ABI Prism 7700 Sequence Detector Instrument (Applied Biosystems, Vienna, Austria). Sequences of primers and probes (3′ FAM, 5′-TAMRA label; synthesized by Microsynth, Balgach, Switzerland) were:

murine IDO1: primer forward, 5′-GGCTTTGCTCTACCACATCCAC-3′; primer reverse, 5′-TAGCCACAAGGACCCAGGG-3′; probe, 5′-CTGTATGCGTCGGGCAGCTCCA-3′;

murine IDO2: primer forward, 5′-TCATGCCTTCGATGAGTTCCT-3′; primer reverse, 5′-GCATGTAGTCCCTCATTCTGTGTAGA-3′; probe, 5′-AGCCAACACTTTCCTTGCAATGCTCAA-3′; 18S RNA: primer forward, 5′-CCATTCGAACGTCTGCCCTAT-3′; primer reverse, 5′-TCACCCGTGGTCACCATG-3′, probe, 5′-ACTTTCGATGGTAGTCGCCGTGCCT-3′; and

murine granzyme B (GZMB): primer forward, 5′-CCCCAATGGGCAAAT-ACTCA-3′; primer reverse, 5′-TCACACTCCCGATCCTTCTGT-3′; probe, 5′-CACGCTACAAGAGGTTGAGCTGACAG-3′; murine FOXP3: primer forward, 5′-TCTACCATTGGTTTACTCGCATGT-3′; primer reverse, 5′-TGGCGGATGGCATTCTTC-3′; probe, 5′-CGCCTACTTCAGAAACCACCCCGC-3′.

Anti-donor Abs

Recipient serum obtained at the day of rejection or day 100 after transplant was heat inactivated and incubated with recipient- and donor-type thymocytes, which are low in Fc-receptors, reducing background. Binding of serum IgG Abs to thymocytes was detected using FITC-conjugated rat anti-mouse IgG1 and IgG2a/2b (BD Biosciences, Schwechat, Austria) and analyzed by flow cytometric analysis.

Statistical analysis

Data were analyzed using Prism 4.0 (GraphPad Software, San Diego, CA). Statistical significance was determined by a Student t test (between two groups) or ANOVA with a post hoc test (three or more groups). Average duration of graft survival presented as the median was analyzed according to the Kaplan–Meier method. The Mantel–Cox test was used to compare survival curves for different subgroups on univariate analyses; p < 0.05 was considered statistically significant.

Results

CTLA4Ig plus DST induces indefinite graft survival in a fully MHC-mismatched murine cardiac transplant model

C57BL/6 (H-2b) mice received fully MHC-mismatched cardiac allografts from BALB/c (H-2d) donors. As shown in Fig. 1A, untreated animals showed a median graft survival time (MST) of 9 d. Treatment with CTLA4Ig led to significant prolongation of graft survival with occasional long-term survival (MST, 41 d). When additional therapy was given in the form of a DST at the time of transplantation, the outcome was further improved substantially, with virtually all animals having indefinite (MST > 100 d) graft survival. Long-term survival observed in these animals was not due to DST alone, because mice receiving DST without CTLA4Ig uniformly rejected their grafts with an MST of 10 d comparable to untreated controls.

FIGURE 1.
FIGURE 1.

DST + CTLA4Ig treatment results in long-term allograft survival. C57BL/6 mice received fully mismatched BALB/c heart allografts. Treatment regimens included: ▪, no immunosuppression (n = 12); ◇, DST (5 × 106 splenocytes i.v.; n = 11); △, CTLA4Ig (500 μg on day 0 and 250 μg on days 2, 4, and 6; n = 11); ○, DST + CTLA4Ig (n = 6). A, MST: ▪, 9 d; ◇, 10 d; △, 41 d; and ○, >100 d (p < 0.0001, Mantel–Cox Test). B, Representative H&E images of heart grafts at either the time of clinical rejection or postoperative day 100 from DST + CTLA4Ig-treated animals (original magnification ×20, inset ×40, zoom ×4). C, ISHLT rejection scores (mean ± SD): no IS, 3.83 ± 0.41; DST, 3.0 ± 0.90; CTLA4Ig, 3.67 ± 0.82; DST + CTLA4Ig, 1.67 ± 1.4. *p < 0.001, ANOVA. IS, immunosuppression.

To further confirm rejection and to examine the condition of cardiac allografts, we performed histopathologic analyses at the time of clinical rejection (loss of palpable heartbeat) or at day 100 after transplantation. Representative images and ISHLT rejection scores are shown in Fig. 1B and 1C. Untreated allografts and those treated with DST or CTLA4Ig alone showed diffuse, perivascular, or interstitial infiltrates with myocyte damage and focal necrosis. In contrast, DST + CTLA4Ig-treated allografts were notable for the excellent preservation of myocardial histology, and they revealed almost no cellular infiltrates on day 100 after transplantation. In addition, DST when added to CTLA4Ig treatment was able to prevent signs of chronic cardiac allograft rejection such as interstitial fibrosis, focal scarring, myointimal proliferation, and transplant arteriosclerosis.

CTLA4Ig increases intragraft IDO expression and enzyme activity

Because the role of IDO-mediated tryptophan catabolism in the development of indefinite graft survival in this vascularized transplant model is unknown, we assessed IDO protein and gene expression to further elucidate the tolerogenic mechanisms of CTLA4Ig.

First, to localize and quantify intragraft expression of IDO, all cardiac transplants were stained for IDO. Quantitative IDO expression was calculated as outlined in Materials and Methods. Representative images and IDO staining scores (mean ± SD) are shown in Fig. 2A and 2B. Untreated controls showed no or minimal IDO expression in the native heart (0.35 ± 0.16), kidney (0.55 ± 0.23), and liver (0.60 ± 0.15) and weak to moderate expression in lymph nodes (0.90 ± 0.28) and spleen (1.10 ± 0.19). In contrast, IDO expression and IDO+ cells were found in all animals upon treatment with CTLA4Ig. However, the highest IDO scores were present in long-term surviving allografts of DST + CTLA4Ig-treated recipients (Fig. 2B). IDO was mainly expressed in vascular endothelial cells and cells morphologically similar to APCs, such as macrophages and DCs identified as larger angular cells. The absolute number of IDO+ cells found in the CTLA4Ig-treated groups was much higher than in grafts from untreated or DST alone treated animals, which showed few or no IDO-expressing cells (Fig. 2B). In addition to the high intragraft IDO expression (1.83 ± 0.75), DST + CTLA4Ig treatment resulted in increased IDO expression and staining scores in lymph node (1.33 ± 0.16) and spleen (1.67 ± 0.17), but did not alter baseline expression in the kidney (0.41 ± 0.20) or liver (0.33 ± 0.17). Nontransplanted animals showed upregulation of IDO while treated with CTLA4-Ig, but no prolonged increase in IDO expression and activity as opposed to transplant recipients (data not shown). We therefore hypothesize that the graft contributes to a prolonged and maintained increased IDO activity after CTLA4-Ig + DST treatment.

FIGURE 2.
FIGURE 2.

CTLA4Ig induces IDO expression and enzyme activity. A, Representative IHC images of intragraft IDO expression are shown in individual treatment groups (original magnification ×20, inset ×40, zoom ×4). B, IDO expression was scored semiquantitatively based on staining intensity and distribution as detailed in Materials and Methods. IHC score: no IS, 0; DST, 0.17 ± 0.41; CTLA4Ig, 0.83 ± 0.75; DST + CTLA4Ig, 1.83 ± 0.75. Data are presented as mean ± SD, *p < 0.0001, ANOVA. C, Quantitative real-time PCR (Taqman Technology) for intragraft IDO1 (no IS, 2.68e-006 ± 1.41e-006; DST, 1.31e-006 ± 1.10e-006; CTLA4Ig, 1.31e-006 ± 1.09e-006; DST + CTLA4Ig, 1.37e-006 ± 1.18e-006) and IDO2 (no IS, 3.10e-008 ± 1.40e-008; DST, 1.80e-008 ± 1.58e-008; CTLA4Ig, 2.59e-008 ± 1.79e-008; DST + CTLA4Ig, 2.99e-008 ± 2.35e-008) expression; IDO1 expression in syngeneic grafts was 1.47e-007 ± 1.05e-007. p = n.s., ANOVA. D, Changes in IDO activity assessed as serum kyn/trp ratio by HPLC. ▪: day 0, 1 ± 0.23; day 5, 0.94 ± 0.33; day 10, 0.51 ± 0.19; ◇: day 0, 1 ± 0.28; day 5, 0.64 ± 0.16; day 10, 0.45 ± 0.11; △: day 0, 1 ± 0.49; day 5, 1.30 ± 0.15; day 10, 0.96 ± 0.48; day 15, 0.72 ± 0.31; day 100, 0.69 ± 0.28; ○: day 0, 1 ± 0.44; day 5, 1.76 ± 0.05; day 10, 1.79 ± 0.24; day 15, 1.63 ± 0.13; day 100, 1.26 ± 0.44. Data are given as mean ± SD. *p < 0.05. IS, immunosuppression.

Notably, qPCR performed for IDO1 and IDO2 showed significant increased levels compared with syngeneic controls, but no significant differences in levels of intragraft gene expression at the day of rejection or endpoint of the study respectively in either experimental group (Fig. 2C). Because these analyses were performed with samples taken either at the time of rejection, or on day 100 if no rejection was seen, the differences in IDO staining scores and expression may reflect the presence of rejection rather than the direct effect of the treatment. However, despite this limitation the data are in agreement with reports of other groups who have demonstrated that IDO expression increases in grafts during both tolerance and rejection (19, 20).

In addition, to transcriptional regulation, IDO function is subject to signals that alter enzyme activity without affecting transcription or translation (21). Therefore, we next examined IDO activity by means of serum-free tryptophan and kynurenine concentrations, as an indirect estimate of IDO activity or metabolism. Fitting with the immunochemistry data, and as shown in Fig. 2D, IDO activity was significantly elevated in the tolerant group receiving CTLA4Ig + DST as early as by day 5 after transplantation when compared with the other regimens. These differences in IDO activity remained significant throughout the entire observation period. Pretransplant kyn/trp levels did not differ between groups.

IDO inhibition with 1-methyl-dl-tryptophan abrogates tolerogenic effects of CTLA4Ig

IDO has been shown in several in vitro studies to inhibit alloreactive immune responses, yet there are limited in vivo data from solid organ transplant models to support this finding. To determine whether IDO activity is required for CTLA4Ig + DST-mediated long-term graft survival, we next treated allograft recipients with 1-methyl-dl-tryptophan (1-MT), a pharmacologic IDO inhibitor. When recipients received 1-MT at the time of transplantation to block IDO activity in vivo, treatment with CTLA4Ig + DST was no longer able to promote long-term graft acceptance, and rejection occurred with median survival times comparable to untreated controls (Fig. 3A). In addition, when recipients with stable and robust graft function received 1-MT delayed at day 50 after transplantation, tolerance was lost and all cardiac grafts rejected promptly (Fig. 3A). Survival data were again confirmed by histologic analysis (Fig. 3B) showing significantly lower ISHLT-scores in grafts from CTLA4Ig + DST-treated animals compared with the other regimens (Fig. 3C).

FIGURE 3.
FIGURE 3.

DST + CTLA4Ig-induced long-term graft acceptance depends on IDO activity. C57BL/6 wt or IDO−/− mice received fully mismatched BALB/c heart allografts. Treatment regimens included (▪) no immunosuppression (n = 12); ○, DST (5 × 106 splenocytes i.v.) + CTLA4Ig (500 μg on day 0 and 250 μg on days 2, 4, and 6; n = 6); ×, DST + CTLA4Ig + 1-MT d0 (200 mg per 9 d, slow release pellets; n = 13); +, DST + CTLA4Ig + 1-MT d50 (200 mg per 9 d, slow release pellets; n = 5); *, DST + CTLA4Ig (IDO−/−; n = 4). A, MST: ▪, 9 d; ○, all grafts > 100 d; ×, 11 d; +, 58 d; *, 18 d. p < 0.001, Mantel–Cox Test. B and C, Representative H&E images and ISHLT rejection scores of heart grafts from individual treatment groups. No IS, 3.83 ± 0.41; DST + CTLA4Ig, 1.67 ± 1.40; DST + CTLA4Ig + 1MT (d 0), 3.71 ± 0.40; DST + CTLA4Ig + 1MT (d 50), 3.75 ± 0.29; DST + CTLA4Ig (IDO−/−), 3.50 ± 0.50. p < 0.001, ANOVA. D, Changes in IDO activity assessed as serum kyn/trp ratio by HPLC. ▪: day 0, 1 ± 0.23; day 5, 0.94 ± 0.33; day 10, 0.51 ± 0.19; ○: day 0, 1 ± 0.44; day 5, 1.76 ± 0.05; day 10, 1.79 ± 0.24; day 15, 1.63 ± 0.13; day 100, 1.26 ± 0.44; ×: day 0, 1 ± 0.28; day 5, 1.78 ± 0.18; day 10, 1.30 ± 0.16; day 15, 1.00; *all days < 0.3. Data are given as mean ± SD. *p < 0.05. IS, immunosuppression.

Systemic IDO enzyme activity of 1-MT–treated animals was significantly lower compared with the CTLA4Ig + DST-treated group (Fig. 3D). Inhibition of IDO activity by 1-MT did not result in significant changes of IDO1 and IDO2 gene expression (data not shown). No effect of control placebo pellets on graft survival was observed (data not shown).

In addition to pharmacologic IDO inhibition, IDO−/− mice lacking a functional IDO1 gene showed also mean graft survival times comparable to control groups following CTLA4Ig + DST treatment (Fig. 3). Systemic IDO activity was almost undetectable (Fig. 3D) and grafts from IDO−/− animals did not reveal expression of IDO1 and IDO2 (data not shown). Nevertheless, slight changes in kynurenine were detectable most likely because of action of tryptophan 2,3-dioxygenase, an additional non–immune-regulated tryptophan-degrading enzyme in the liver.

These in vivo results demonstrate that IDO activity is necessary for both the early induction of long-term allograft survival by CTLA4Ig + DST and for the maintenance phase of graft acceptance mediated by this regimen.

Linked immunomodulation of CTLA4Ig and Tregs

We next investigated the contribution of Tregs toward CTLA4Ig-induced long-term graft survival in this model and assessed expression of the Treg-specific transcription factor Foxp3 by means of immunohistochemistry (IHC) and qPCR. Because Foxp3 expression is unique to Tregs, the presence of Tregs in cardiac allografts should be mirrored by expression of Foxp3. As shown in Fig. 4A and 4B, all CTLA4Ig-treated cardiac grafts displayed significantly higher numbers of Foxp3+ Tregs compared with grafts without costimulatory blocker treatment (Fig. 4A–C). In addition, nodular infiltrates of Foxp3+ cells were often associated with IDO-positive arterial endothelium. Of note, staining for Foxp3 in grafts from IDO−/− animals despite treatment with CTLA4Ig + DST was rare and revealed scores comparable to untreated controls or animals treated with DST alone (Fig. 4A, 4B).

FIGURE 4.
FIGURE 4.

CTLA4Ig increases intragraft Foxp3+ expression. A, Representative IHC images showing intragraft Foxp3 expression in individual treatment groups (original magnification ×20, inset ×40, zoom ×4). B, Foxp3 expression was scored semiquantitatively based on staining intensity and distribution as detailed in Materials and Methods. IHC score: no IS, 0.50 ± 0.05; DST, 0.83 ± 0.41; CTLA4Ig, 1.67 ± 0.52; DST + CTLA4Ig, 1.50 ± 0.84; DST + CTLA4Ig + 1-MT, 1.58 ± 0.51; DST + CTLA4Ig (IDO−/−), 0.66 ± 0.51. Data are presented as mean ± SD. *p < 0.01, ANOVA. C, Quantitative real-time PCR (Taqman technology) for intragraft Foxp3 expression: no IS, 6.55e-007 ± 3.63e-007; DST, 8.18e-007 ± 3.24e-007; CTLA4Ig, 1.67e-006 ± 3.94e-007; DST + CTLA4Ig, 4.01e-006 ± 8.72e-007; DST + CTLA4Ig + 1-MT, 1.45e-006 ± 6.89e-007; DST + CTLA4Ig (IDO−/−), 3.05e-007 ± 4.07e-008. D, Intragraft Foxp3/GZMB ratio assessed by qPCR was significantly higher after DST + CTLA4Ig (0.48 ± 0.14) treatment as compared with the other regimens (no IS, 0.015 ± 0.004; DST, 0.025 ± 0.009; and CTLA4Ig, 0.204 ± 0.066); p < 0.01, ANOVA. IS, immunosuppression.

In addition to IHC staining, total RNA was isolated from cardiac allografts to evaluate for presence or absence of Foxp3 expression. As summarized in Fig. 4C, Foxp3 mRNA was significantly elevated in CTLA4Ig + DST-treated allografts, but was comparable to background and controls in the other groups, with only a slight increase in CTLA4Ig and CTLA4Ig + DST + 1MT-treated grafts.

To further investigate the hypothesis that Foxp3+ cells are an integral component of CTLA4Ig + DST-treated grafts and are dominant over pathogenic effectors, we assessed the intragraft ratio between cytopathic GZMB expressing effector cells and Foxp3+ protective Tregs. Foxp3/GZMB gene expression ratio was significantly higher in the CTLA4Ig + DST group compared with the other regimens (Fig. 4D). These data suggest that there is an increased load in intragraft tolerogenic Tregs upon CTLA4Ig + DST treatment.

Tregs are required for CTLA4Ig-mediated indefinite graft survival

Regarding the mechanisms of induction of indefinite graft survival in this model, the observed intragraft accumulation of Foxp3+ Tregs and the high expression of IDO by graft endothelial cells and DCs indicates an interplay between these two immunoregulatory mechanisms. To test this hypothesis, we next depleted recipient Treg pretransplant using an anti-CD25 mAb that resulted in a loss of indefinite graft survival and survival times decreased to those of untreated controls (Fig. 5A–C). These data demonstrate that following selective pretransplant depletion of Tregs despite unaltered high intragraft expression of IDO, CTLA4Ig + DST was unable to induce long-term graft survival in our model (Fig. 5E). Most interestingly, recipients treated with anti-CD25mAb showed considerably less systemic IDO activity as compared with non–Treg-depleted CTLA4Ig + DST-treated animals (Fig. 5F). In addition, selective late Treg depletion at postoperative day 50 resulted in a loss of indefinite graft survival and rejection following aCD25mAb treatment (Fig. 5A). IDO activity significantly dropped after delayed Treg depletion, and intragraft IDO expression was found to be significantly decreased (Fig. 5E, 5F).

FIGURE 5.
FIGURE 5.

CTLA4Ig-mediated indefinite graft survival requires Tregs. Tregs were depleted in recipient animals using anti-CD25 mAb (500 μg i.p. on day −5) prior to DST + CTLA4Ig treatment to test for their requirement for permanent graft acceptance in this model. A, Recipients (▴) rejected their grafts with an MST of 12 d comparable to untreated controls (p = n.s.). In addition, late depletion of Tregs at posttransplant day 50 (d + 50) in CTLA4Ig + DST treated recipients resulted in acute rejection and an MST of 63 d (▪). B, Representative H&E and Foxp3 IHC images of heart grafts from individual treatment groups (original magnification ×20, inset ×40, zoom ×4). C, ISHLT scores: no IS, 3.83 ± 0.41; DST + CTLA4Ig, 1.67 ± 1.40; DST + CTLA4Ig + aCD25mAb (d −5), 3.60 ± 0.42; DST + CTLA4Ig + aCD25mAB (day +50), 3.20 ± 0.76. D, Foxp3 IHC expression scores: no IS, 0.50 ± 0.50; DST + CTLA4Ig, 1.50 ± 0.84; DST + CTLA4Ig + aCD25mAb (day −5), 0.33 ± 0.21; DST + CTLA4Ig + aCD25mAB (day + 50), 0.42 ± 0.20. E, IDO IHC expression scores: no IS, 0; DST + CTLA4Ig, 1.83 ± 0.30; DST + CTLA4Ig + aCD25mAb (day −5): 1.83 ± 0.60; DST + CTLA4Ig + aCD25mAB (day +50), 0.25 ± 0.17. F, IDO activity assessed as serum kyn/trp ratio by HPLC in DST + CTLA4Ig + aCD25mAb (day −5) was statistically not significant different from (▪). IDO activity in DST + CTLA4Ig + aCD25mAB (day +50) in contrast showed a significant decrease after Treg depletion as compared with ○. Data in C, D, and E are presented as mean ± SD; all p < 0.01, ANOVA. IS, immunosuppression.

CTLA4Ig prevents formation of donor-reactive alloantibodies

It has been previously shown that donor-reactive alloantibodies (DSAs) can be generated by long-term renal allograft-accepting mice at levels comparable to rejecting heart graft recipients (22). We therefore also tested in the current study for alloantibody persistence and the existence of DSA in serum of recipient animals. However, none, except one animal in the CTLA4Ig-alone group developed DSAs (Fig. 6). This finding is in line with early findings by Linsley et al. (23), who demonstrated that costimulation via B7 is essential for humoral responses to occur and that administration of CTLA4Ig suppressed Ab production. CTLA4Ig also prevented the production of alloantibodies in a nonhuman primate model for islet transplantation and has been shown to prevent development of chronic rejection (24).

FIGURE 6.
FIGURE 6.

Donor-reactive alloantibodies are not induced in DST + CTLA4Ig treated recipients. Recipient serum obtained at the day of rejection or postoperative day 100 in long-term survivors was incubated with recipient- and donor-type thymocytes and analyzed by flow cytometry. Whereas primed B6 control mice showed strong Ab responses (49%), except for one animal in the CTLA4Ig-treated group showed weak DSA formation (2.17%) in either study group.

Discussion

This study demonstrates for the first time, to our knowledge, mechanistic in vivo evidence that the development of immune regulation and tolerance by CTLA4Ig + DST toward vascularized cardiac allografts requires and depends on a concerted interplay of both IDO activity and Tregs in addition to costimulatory blockade. It thereby appears, in contrast to previous studies, that CTLA4Ig does not directly activate IDO but induces Tregs that require IDO to facilitate long-term graft acceptance.

Multiple groups have demonstrated potent immunosuppressive properties of CTLA4Ig in vivo using various rodent models for transplantation and autoimmunity, and results from this study confirm previous data showing that CTLA4Ig + DST is a potent and promising means to induce long-term graft survival in fully MHC-mismatched cardiac allografts (23, 25–28).

However, despite the fact that CTLA4 can attenuate T cell function by mainly three distinct mechanisms: 1) because of its higher avidity to B7 family members (CD80 and CD86) than CD28, CTLA4 can scavenge B7 molecules and thereby prevent them from ligating CD28 (2); 2) CTLA4 can actively inhibit TCR-mediated signals; and 3) via reverse signaling into B7-expressing APCs, CTLA4 can induce DC activation, IFN-γ production, and finally IDO enzymatic activity; the exact tolerogenic mechanisms of CTLA4Ig still remain unclear. A similar mechanism to surface CTLA4 has been recently shown for a custom-made form of a CTLA4Ig fusion protein in the immunologic specific setting of islet transplantation, which lead to transcriptional regulation and expression of IDO through the ligation of cell-surface CD80/CD86 molecules (6). In contrast, in the current study using a vascularized solid organ transplant model as well as the clinically available CTLA4Ig Ab abatacept, we found significantly increased IDO activity only in recipients receiving CTLA4Ig + DST, whereas CTLA4Ig alone did not induce IDO and showed only a trend toward increased enzyme activity compared with syngeneic controls. This finding suggests that CTLA4Ig in this model does not induce long-term graft survival and is not able to directly induce IDO.

Localized tryptophan depletion owing to IDO is important in inhibiting T cell proliferation and specific tryptophan catabolites in the kynurenine metabolic pathway act as potent proapoptotic agents in T cells (29). However, despite highly increased IDO expression in CTLA4Ig + DST-treated animals, we did not observe an increased rate of apoptosis in these grafts. Our data instead indicate that IDO functions as a downstream mediator for certain tolerogenic effects of CTLA4Ig. In addition to costimulatory blockade and delivering inhibitory signals to the T cell, CTLA4Ig also induced significant intragraft expression and activation of IDO. Subsequently, IDO finally generates an immunoregulatory microenvironment because of tryptophan depletion and production of kynurenine metabolites; IDO also changes DCs to acquire characteristics that no longer support T cell activation and inhibit T cell proliferation (30, 31). Thus, IDO-mediated tryptophan catabolism induced by CTLA4Ig results in an intragraft milieu that is poor in tryptophan, limiting T cell growth and proliferation, and contributes to the immunoregulatory effects of CTLA4Ig in addition to simple blockade of CD28.

The localization of IDO expression is known to be critical for its immunoregulatory effects. Guillonneau et al. (32) showed that when adenoviral-mediated IDO gene transfer was performed in the graft, survival was significantly prolonged, whereas rejection was not inhibited when IDO gene transfer was performed at a distant site. In addition, Thebault et al. (33) recently showed a key role for IFN-γ and IDO in the induction of local immune privilege in allograft tolerance with an interplay between CD4+CD25+Foxp3+ regulatory T cells and graft endothelial cells, whereas Beutelspacher et al. (34) demonstrated that IDO expression in human endothelial cells (EC) inhibits allogeneic T cell responses and induces anergy in alloreactive T cells. In line with these results, we found IDO expression and IDO+ cells mainly in vascular ECs and APCs such as macrophages and DCs within the graft upon CTLA4Ig treatment. These results further suggest an Ag-specific localized immunoregulatory phenomenon for CTLA4Ig-induced IDO rather than a nonspecific immunosuppression.

The role of cytokines in the induction of graft acceptance by means of costimulatory blockade is complex and still remains incompletely understood. IFN-γ plays an important role in some CTLA4Ig-induced tolerance models, and it has been previously shown that IFN-γ is required to permit the graft-prolonging effect of CTLA4Ig + anti-CD154 to occur in skin and heart graft models (35). Moreover, IFN-γ has certain anti-inflammatory and tolerogenic roles, in that it enables induced Tregs to control alloimmune responses and that transplantation tolerance cannot be induced using costimulation blockade in IFN-γ knockout mice (35, 36). However, further investigation is suggested to determine whether these critical effects of IFN-γ toward CTLA4Ig-induced tolerance are mediated via its potent effects as the main activator of IDO.

Data from this study also demonstrate that IDO activity is required for both the early induction of long-term cardiac allograft survival by CTLA4Ig + DST and for the maintenance phase of indefinite graft survival. In line with these results, IDO has been demonstrated to be critical to maintain graft survival in various models of spontaneous liver and kidney tolerance (37, 38). Laurence et al. (19) found an increase in IDO expression in grafts of spontaneously accepted rat livers and that long-term graft survival was prevented by blocking IDO in the early posttransplant period. Similar results were reported by Miki et al. (39) in a mouse model of liver tolerance. Furthermore, spontaneous renal allograft acceptance was shown to evolve through a series of mechanisms, including high intragraft IDO expression, regulatory DCs, and Tregs, which combined facilitated robust tolerogenic immune regulation (37).

Recently Tregs have been implicated in tolerance mechanisms involved in solid organ transplant models using treatment with CTLA4Ig and evidence for the in vivo generation of Tregs by CTLA4Ig has been hypothesized (14, 40). However, it is still unknown whether the immunosuppressive effects of CTLA4Ig on T cell alloresponses in the setting of vascularized solid organ transplants are mediated at least in part through the generation of Tregs. The finding of significantly increased Foxp3 levels and nodular infiltrates of Foxp3+ cells associated with IDO-positive arterial endothelium in CTLA4Ig + DST-treated grafts suggests that, to some extend, the mechanism of action of CTLA4Ig is closely linked and operates in Tregs in this model. Activated Tregs upregulate CTLA4 surface expression above the constitutive levels normally expressed by these cells, and exposure of activated Tregs to DCs has been shown to result in IFN-γ and IDO production by the DCs (2, 14, 30). In contrast, B7-CD28 signals are also important for the homeostasis of CD4+CD25+ Tregs, and prolonged CTLA4Ig treatment has been shown to lead to depletion of Tregs because of decreased IL-2 levels (16, 41). This might not be the case in the current study because only a short course of CTLA4Ig + DST was used to induce indefinite graft survival in this protocol.

Most of these data suggesting a protolerogenic role of IDO were generated in murine models. Whether similar DC subsets and immunoregulatory mechanisms are also in effect in human DCs still remains controversial. Vacca et al. (42) recently showed that matured human DCs are refractory to induction of IDO by CTLA4Ig and do not inhibit T cell responses. Similarly, studies by Terness et al. and Löb et al. (43–45) showed that human DCs do not suppress T cell responses even if rendered IDO positive with IFN-γ.

Strikingly, Foxp3 staining and expression in grafts from IDO−/− animals, despite treatment with CTLA4Ig + DST, was rare and revealed scores similar to untreated controls, whereas pharmacologic inhibition of IDO with 1-MT did not impair the number of Foxp3+ Tregs. This finding might argue for a constitutive requirement of IDO for de novo generation of Tregs induced by CTLA4Ig that warrants further investigation.

In addition, previous data have shown that Tregs are not only present in lymphoid organs, such as lymph node and spleen; they also infiltrate tolerated allografts where they hold effector cells in check and further reinforce a state of dominant tolerance (46). The high proportion of Foxp3+ Tregs found in the grafts of long-term surviving animals in this study also indicates that these cells are actively recruited to or even might be expanded at this site, and owing to their constitutively high expression of CTLA4 can induce expression of IDO in DCs/ECs and promote tolerance via regulatory feedback loops. This finding is also substantiated by the fact that late depletion of Tregs with aCD25mAbs in recipients with long-term surviving grafts resulted in acute rejection and graft loss paralleled by a significant decrease in IDO expression and function. Furthermore, the finding of a significantly increased Foxp3/GZMB gene expression ratio in the CTLA4Ig + DST group suggests that there is an increased load in intragraft tolerogenic Tregs and fits well with previous studies in skin and islet transplant models showing a favorable balance of Foxp3/GZMB ratios toward a regulatory Foxp3+ Treg-dominated profile (47, 48).

As a result, Tregs may be instrumental within the graft to facilitate a concerted immunoregulatory mechanism involving IDO to induce indefinite graft survival and are not only an epiphenomenon of this model. This dual IDO and Treg hypothesis for CTLA4Ig-induced long-term graft survival is also not mutually exclusive, and there is more emerging data that suggest that there is a synergistic, complementary relationship between the two (2, 49). In fact, Tregs have been demonstrated to interact with DCs to initiate IDO expression, and conversely it has been shown that IDO+ DCs are capable of stimulating the development of Tregs (33, 50, 51). Immunohistochemistry data in this study clearly demonstrate both Foxp3 and IDO expression in accepted allografts, which is consistent with such a hypothesis of synergism. Similar results have been found recently in a model of spontaneous renal allograft acceptance by Cook et al. (37), and Thebault et al. (33) showed in a cardiac allograft model that such cells accumulate in the graft and induce endothelial cell expression of IDO after transfer of Foxp3+ Tregs to a secondary irradiated recipient. Our data, however, extend previous findings and suggest that CTLA4Ig initiates and facilitates the interplay between IDO+ cells, Tregs, and effector cells within the graft microenvironment leading to local immune privilege and long-term graft acceptance.

In conclusion, this study reveals that the development of immune regulation and indefinite acceptance by CTLA4Ig + DST toward vascularized cardiac allografts in addition to costimulatory blockade depends on both Tregs and IDO. CTLA4Ig thereby does not directly activate IDO but induces Foxp3+ Tregs that require IDO to mediate their regulatory protolerogenic effects. In addition, these Tregs may serve to amplify or sustain intragraft IDO activity.

Disclosures

The authors have no financial conflicts of interest.

Acknowledgments

We thank Maria Gleisner, Astrid Haara, Petra Loitzl, and Nina Madl for expert technical assistance.

Footnotes

  • This work was supported by Jubiläumsfonds der Österreichischen Nationalbank Project 12239 (to G.B.) and by the Austrian Science Fund zur Förderung der wissenschaftlichen Forschung Project 22289 (to E.R.W.).

  • Abbreviations used in this article:

    CTLA4Ig
    cytotoxic T lymphocyte-associated Ag-4 Ig
    DC
    dendritic cell
    DSA
    donor-reactive alloantibodies
    DST
    donor-specific transfusion
    EC
    endothelial cell
    GZMB
    granzyme B
    IHC
    immunohistochemistry
    ISHLT
    International Society for Heart and Lung Transplantation
    kyn/trp
    kynurenine/tryptophan
    MST
    median graft survival time
    1-MT
    1-methyl-dl-tryptophan
    qPCR
    qantitative PCR
    Treg
    regulatory T cell.

  • Received August 16, 2010.
  • Accepted October 18, 2011.

References

  1. ↵
    1. Lechler R. I.,
    2. M. Sykes,
    3. A. W. Thomson,
    4. L. A. Turka
    . 2005. Organ transplantation—how much of the promise has been realized? Nat. Med. 11: 605–613.
    OpenUrlCrossRefPubMed
  2. ↵
    1. Alegre M. L.,
    2. F. Fallarino
    . 2006. Mechanisms of CTLA-4-Ig in tolerance induction. Curr. Pharm. Des. 12: 149–160.
    OpenUrlCrossRefPubMed
  3. ↵
    1. Larsen C. P.,
    2. E. T. Elwood,
    3. D. Z. Alexander,
    4. S. C. Ritchie,
    5. R. Hendrix,
    6. C. Tucker-Burden,
    7. H. R. Cho,
    8. A. Aruffo,
    9. D. Hollenbaugh,
    10. P. S. Linsley,
    11. et al
    . 1996. Long-term acceptance of skin and cardiac allografts after blocking CD40 and CD28 pathways. Nature 381: 434–438.
    OpenUrlCrossRefPubMed
  4. ↵
    1. Kirk A. D.,
    2. D. M. Harlan,
    3. N. N. Armstrong,
    4. T. A. Davis,
    5. Y. Dong,
    6. G. S. Gray,
    7. X. Hong,
    8. D. Thomas,
    9. J. H. Fechner Jr..,
    10. S. J. Knechtle
    . 1997. CTLA4-Ig and anti-CD40 ligand prevent renal allograft rejection in primates. Proc. Natl. Acad. Sci. USA 94: 8789–8794.
    OpenUrlAbstract/FREE Full Text
  5. ↵
    1. Vincenti F.,
    2. C. Larsen,
    3. A. Durrbach,
    4. T. Wekerle,
    5. B. Nashan,
    6. G. Blancho,
    7. P. Lang,
    8. J. Grinyo,
    9. P. F. Halloran,
    10. K. Solez,
    11. et al,
    12. Belatacept Study Group
    . 2005. Costimulation blockade with belatacept in renal transplantation. N. Engl. J. Med. 353: 770–781.
    OpenUrlCrossRefPubMed
  6. ↵
    1. Grohmann U.,
    2. C. Orabona,
    3. F. Fallarino,
    4. C. Vacca,
    5. F. Calcinaro,
    6. A. Falorni,
    7. P. Candeloro,
    8. M. L. Belladonna,
    9. R. Bianchi,
    10. M. C. Fioretti,
    11. P. Puccetti
    . 2002. CTLA-4-Ig regulates tryptophan catabolism in vivo. Nat. Immunol. 3: 1097–1101.
    OpenUrlCrossRefPubMed
  7. ↵
    1. Mellor A. L.,
    2. B. Baban,
    3. P. Chandler,
    4. B. Marshall,
    5. K. Jhaver,
    6. A. Hansen,
    7. P. A. Koni,
    8. M. Iwashima,
    9. D. H. Munn
    . 2003. Cutting edge: induced indoleamine 2,3 dioxygenase expression in dendritic cell subsets suppresses T cell clonal expansion. J. Immunol. 171: 1652–1655.
    OpenUrlAbstract/FREE Full Text
    1. Mellor A. L.,
    2. P. Chandler,
    3. B. Baban,
    4. A. M. Hansen,
    5. B. Marshall,
    6. J. Pihkala,
    7. H. Waldmann,
    8. S. Cobbold,
    9. E. Adams,
    10. D. H. Munn
    . 2004. 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.
    OpenUrlAbstract/FREE Full Text
  9. ↵
    1. Baban B.,
    2. A. M. Hansen,
    3. P. R. Chandler,
    4. A. Manlapat,
    5. A. Bingaman,
    6. D. J. Kahler,
    7. D. H. Munn,
    8. A. L. Mellor
    . 2005. A minor population of splenic dendritic cells expressing CD19 mediates IDO-dependent T cell suppression via type I IFN signaling following B7 ligation. Int. Immunol. 17: 909–919.
    OpenUrlAbstract/FREE Full Text
  10. ↵
    1. Brandacher G.,
    2. R. Margreiter,
    3. D. Fuchs
    . 2007. Implications of IFN-gamma-mediated tryptophan catabolism on solid organ transplantation. Curr. Drug Metab. 8: 273–282.
    OpenUrlCrossRefPubMed
    1. Liu X.,
    2. N. Shin,
    3. H. K. Koblish,
    4. G. Yang,
    5. Q. Wang,
    6. K. Wang,
    7. L. Leffet,
    8. M. J. Hansbury,
    9. B. Thomas,
    10. M. Rupar,
    11. et al
    . 2010. Selective inhibition of IDO1 effectively regulates mediators of antitumor immunity. Blood 115: 3520–3530.
    OpenUrlAbstract/FREE Full Text
    1. Hainz U.,
    2. P. Obexer,
    3. C. Winkler,
    4. P. Sedlmayr,
    5. O. Takikawa,
    6. H. Greinix,
    7. A. Lawitschka,
    8. U. Pötschger,
    9. D. Fuchs,
    10. S. Ladisch,
    11. A. Heitger
    . 2005. Monocyte-mediated T-cell suppression and augmented monocyte tryptophan catabolism after human hematopoietic stem-cell transplantation. Blood 105: 4127–4134.
    OpenUrlAbstract/FREE Full Text
  13. ↵
    1. Curti A.,
    2. S. Trabanelli,
    3. V. Salvestrini,
    4. M. Baccarani,
    5. R. M. Lemoli
    . 2009. The role of indoleamine 2,3-dioxygenase in the induction of immune tolerance: focus on hematology. Blood 113: 2394–2401.
    OpenUrlAbstract/FREE Full Text
  14. ↵
    1. Fallarino F.,
    2. U. Grohmann,
    3. K. W. Hwang,
    4. C. Orabona,
    5. C. Vacca,
    6. R. Bianchi,
    7. M. L. Belladonna,
    8. M. C. Fioretti,
    9. M. L. Alegre,
    10. P. Puccetti
    . 2003. Modulation of tryptophan catabolism by regulatory T cells. Nat. Immunol. 4: 1206–1212.
    OpenUrlCrossRefPubMed
  15. ↵
    1. Jürgens B.,
    2. U. Hainz,
    3. D. Fuchs,
    4. T. Felzmann,
    5. A. Heitger
    . 2009. Interferon-gamma-triggered indoleamine 2,3-dioxygenase competence in human monocyte-derived dendritic cells induces regulatory activity in allogeneic T cells. Blood 114: 3235–3243.
    OpenUrlAbstract/FREE Full Text
  16. ↵
    1. Wekerle T.,
    2. J. Kurtz,
    3. S. Bigenzahn,
    4. Y. Takeuchi,
    5. M. Sykes
    . 2002. Mechanisms of transplant tolerance induction using costimulatory blockade. Curr. Opin. Immunol. 14: 592–600.
    OpenUrlCrossRefPubMed
  17. ↵
    1. Laich A.,
    2. G. Neurauter,
    3. B. Widner,
    4. D. Fuchs
    . 2002. More rapid method for simultaneous measurement of tryptophan and kynurenine by HPLC. Clin. Chem. 48: 579–581.
    OpenUrlFREE Full Text
  18. ↵
    1. Brandacher G.,
    2. A. Perathoner,
    3. R. Ladurner,
    4. S. Schneeberger,
    5. P. Obrist,
    6. C. Winkler,
    7. E. R. Werner,
    8. G. Werner-Felmayer,
    9. H. G. Weiss,
    10. G. Göbel,
    11. et al
    . 2006. Prognostic value of indoleamine 2,3-dioxygenase expression in colorectal cancer: effect on tumor-infiltrating T cells. Clin. Cancer Res. 12: 1144–1151.
    OpenUrlAbstract/FREE Full Text
  19. ↵
    1. Laurence J. M.,
    2. C. Wang,
    3. E. T. Park,
    4. A. Buchanan,
    5. A. Clouston,
    6. R. D. Allen,
    7. G. W. Mccaughan,
    8. G. A. Bishop,
    9. A. F. Sharland
    . 2008. Blocking indoleamine dioxygenase activity early after rat liver transplantation prevents long-term survival but does not cause acute rejection. Transplantation 85: 1357–1361.
    OpenUrlCrossRefPubMed
  20. ↵
    1. Beutelspacher S. C.,
    2. R. Pillai,
    3. M. P. Watson,
    4. P. H. Tan,
    5. J. Tsang,
    6. M. O. McClure,
    7. A. J. George,
    8. D. F. Larkin
    . 2006. Function of indoleamine 2,3-dioxygenase in corneal allograft rejection and prolongation of allograft survival by over-expression. Eur. J. Immunol. 36: 690–700.
    OpenUrlCrossRefPubMed
  21. ↵
    1. Thomas S. R.,
    2. H. Salahifar,
    3. R. Mashima,
    4. N. H. Hunt,
    5. D. R. Richardson,
    6. R. Stocker
    . 2001. Antioxidants inhibit indoleamine 2,3-dioxygenase in IFN-gamma-activated human macrophages: posttranslational regulation by pyrrolidine dithiocarbamate. J. Immunol. 166: 6332–6340.
    OpenUrlAbstract/FREE Full Text
  22. ↵
    1. Bickerstaff A. A.,
    2. J. J. Wang,
    3. R. P. Pelletier,
    4. C. G. Orosz
    . 2001. Murine renal allografts: spontaneous acceptance is associated with regulated T cell-mediated immunity. J. Immunol. 167: 4821–4827.
    OpenUrlAbstract/FREE Full Text
  23. ↵
    1. Linsley P. S.,
    2. P. M. Wallace,
    3. J. Johnson,
    4. M. G. Gibson,
    5. J. L. Greene,
    6. J. A. Ledbetter,
    7. C. Singh,
    8. M. A. Tepper
    . 1992. Immunosuppression in vivo by a soluble form of the CTLA-4 T cell activation molecule. Science 257: 792–795.
    OpenUrlAbstract/FREE Full Text
  24. ↵
    1. Adams A. B.,
    2. N. Shirasugi,
    3. M. M. Durham,
    4. E. Strobert,
    5. D. Anderson,
    6. P. Rees,
    7. S. Cowan,
    8. H. Xu,
    9. Y. Blinder,
    10. M. Cheung,
    11. et al
    . 2002. Calcineurin inhibitor-free CD28 blockade-based protocol protects allogeneic islets in nonhuman primates. Diabetes 51: 265–270.
    OpenUrlAbstract/FREE Full Text
  25. ↵
    1. Lin H.,
    2. S. F. Bolling,
    3. P. S. Linsley,
    4. R. Q. Wei,
    5. D. Gordon,
    6. C. B. Thompson,
    7. L. A. Turka
    . 1993. Long-term acceptance of major histocompatibility complex mismatched cardiac allografts induced by CTLA4Ig plus donor-specific transfusion. J. Exp. Med. 178: 1801–1806.
    OpenUrlAbstract/FREE Full Text
    1. Akalin E.,
    2. A. Chandraker,
    3. M. E. Russell,
    4. L. A. Turka,
    5. W. W. Hancock,
    6. M. H. Sayegh
    . 1996. CD28-B7 T cell costimulatory blockade by CTLA4Ig in the rat renal allograft model: inhibition of cell-mediated and humoral immune responses in vivo. Transplantation 62: 1942–1945.
    OpenUrlCrossRefPubMed
    1. Shiraishi T.,
    2. Y. Yasunami,
    3. M. Takehara,
    4. T. Uede,
    5. K. Kawahara,
    6. T. Shirakusa
    . 2002. Prevention of acute lung allograft rejection in rat by CTLA4Ig. Am. J. Transplant. 2: 223–228.
    OpenUrlCrossRefPubMed
  28. ↵
    1. Bolling S. F.,
    2. H. Lin,
    3. R. Q. Wei,
    4. L. A. Turka
    . 1996. Preventing allograft rejection with CTLA4IG: effect of donor-specific transfusion route or timing. J. Heart Lung Transplant. 15: 928–935.
    OpenUrlPubMed
  29. ↵
    1. Fallarino F.,
    2. U. Grohmann,
    3. C. Vacca,
    4. R. Bianchi,
    5. C. Orabona,
    6. A. Spreca,
    7. M. C. Fioretti,
    8. P. Puccetti
    . 2002. T cell apoptosis by tryptophan catabolism. Cell Death Differ. 9: 1069–1077.
    OpenUrlCrossRefPubMed
  30. ↵
    1. Mellor A. L.,
    2. D. H. Munn
    . 2004. IDO expression by dendritic cells: tolerance and tryptophan catabolism. Nat. Rev. Immunol. 4: 762–774.
    OpenUrlCrossRefPubMed
  31. ↵
    1. Orabona C.,
    2. P. Puccetti,
    3. C. Vacca,
    4. S. Bicciato,
    5. A. Luchini,
    6. F. Fallarino,
    7. R. Bianchi,
    8. E. Velardi,
    9. K. Perruccio,
    10. A. Velardi,
    11. et al
    . 2006. Toward the identification of a tolerogenic signature in IDO-competent dendritic cells. Blood 107: 2846–2854.
    OpenUrlAbstract/FREE Full Text
  32. ↵
    1. Guillonneau C.,
    2. M. Hill,
    3. F. X. Hubert,
    4. E. Chiffoleau,
    5. C. Hervé,
    6. X. L. Li,
    7. M. Heslan,
    8. C. Usal,
    9. L. Tesson,
    10. S. Ménoret,
    11. et al
    . 2007. CD40Ig treatment results in allograft acceptance mediated by CD8CD45RC T cells, IFN-gamma, and indoleamine 2,3-dioxygenase. J. Clin. Invest. 117: 1096–1106.
    OpenUrlCrossRefPubMed
  33. ↵
    1. Thebault P.,
    2. T. Condamine,
    3. M. Heslan,
    4. M. Hill,
    5. I. Bernard,
    6. A. Saoudi,
    7. R. Josien,
    8. I. Anegon,
    9. M. C. Cuturi,
    10. E. Chiffoleau
    . 2007. Role of IFNgamma in allograft tolerance mediated by CD4+CD25+ regulatory T cells by induction of IDO in endothelial cells. Am. J. Transplant. 7: 2472–2482.
    OpenUrlCrossRefPubMed
  34. ↵
    1. Beutelspacher S. C.,
    2. P. H. Tan,
    3. M. O. McClure,
    4. D. F. Larkin,
    5. R. I. Lechler,
    6. A. J. George
    . 2006. Expression of indoleamine 2,3-dioxygenase (IDO) by endothelial cells: implications for the control of alloresponses. Am. J. Transplant. 6: 1320–1330.
    OpenUrlCrossRefPubMed
  35. ↵
    1. Konieczny B. T.,
    2. Z. Dai,
    3. E. T. Elwood,
    4. S. Saleem,
    5. P. S. Linsley,
    6. F. K. Baddoura,
    7. C. P. Larsen,
    8. T. C. Pearson,
    9. F. G. Lakkis
    . 1998. IFN-gamma is critical for long-term allograft survival induced by blocking the CD28 and CD40 ligand T cell costimulation pathways. J. Immunol. 160: 2059–2064.
    OpenUrlAbstract/FREE Full Text
  36. ↵
    1. Wood K. J.,
    2. B. Sawitzki
    . 2006. Interferon gamma: a crucial role in the function of induced regulatory T cells in vivo. Trends Immunol. 27: 183–187.
    OpenUrlCrossRefPubMed
  37. ↵
    1. Cook C. H.,
    2. A. A. Bickerstaff,
    3. J. J. Wang,
    4. T. Nadasdy,
    5. P. Della Pelle,
    6. R. B. Colvin,
    7. C. G. Orosz
    . 2008. Spontaneous renal allograft acceptance associated with “regulatory” dendritic cells and IDO. J. Immunol. 180: 3103–3112.
    OpenUrlAbstract/FREE Full Text
  38. ↵
    1. Lin Y. C.,
    2. C. L. Chen,
    3. T. Nakano,
    4. S. Goto,
    5. Y. H. Kao,
    6. L. W. Hsu,
    7. C. Y. Lai,
    8. B. Jawan,
    9. Y. F. Cheng,
    10. C. Tateno,
    11. K. Yoshizato
    . 2008. Immunological role of indoleamine 2,3-dioxygenase in rat liver allograft rejection and tolerance. J. Gastroenterol. Hepatol. 23: e243–e250.
    OpenUrlCrossRefPubMed
  39. ↵
    1. Miki T.,
    2. H. Sun,
    3. Y. Lee,
    4. A. Tandin,
    5. A. M. Kovscek,
    6. V. Subbotin,
    7. J. J. Fung,
    8. L. A. Valdivia
    . 2001. Blockade of tryptophan catabolism prevents spontaneous tolerogenicity of liver allografts. Transplant. Proc. 33: 129–130.
    OpenUrlCrossRefPubMed
  40. ↵
    1. Lee R. S.,
    2. J. R. Rusche,
    3. M. E. Maloney,
    4. D. H. Sachs,
    5. M. H. Sayegh,
    6. J. C. Madsen
    . 2001. CTLA4Ig-induced linked regulation of allogeneic T cell responses. J. Immunol. 166: 1572–1582.
    OpenUrlAbstract/FREE Full Text
  41. ↵
    1. Salomon B.,
    2. D. J. Lenschow,
    3. L. Rhee,
    4. N. Ashourian,
    5. B. Singh,
    6. A. Sharpe,
    7. J. A. Bluestone
    . 2000. B7/CD28 costimulation is essential for the homeostasis of the CD4+CD25+ immunoregulatory T cells that control autoimmune diabetes. Immunity 12: 431–440.
    OpenUrlCrossRefPubMed
  42. ↵
    1. Vacca C.,
    2. F. Fallarino,
    3. K. Perruccio,
    4. C. Orabona,
    5. R. Bianchi,
    6. S. Gizzi,
    7. A. Velardi,
    8. M. C. Fioretti,
    9. P. Puccetti,
    10. U. Grohmann
    . 2005. CD40 ligation prevents onset of tolerogenic properties in human dendritic cells treated with CTLA-4-Ig. Microbes Infect. 7: 1040–1048.
    OpenUrlCrossRefPubMed
  43. ↵
    1. Terness P.,
    2. J. J. Chuang,
    3. T. Bauer,
    4. L. Jiga,
    5. G. Opelz
    . 2005. Regulation of human auto- and alloreactive T cells by indoleamine 2,3-dioxygenase (IDO)-producing dendritic cells: too much ado about IDO? Blood 105: 2480–2486.
    OpenUrlAbstract/FREE Full Text
    1. Terness P.,
    2. J. J. Chuang,
    3. G. Opelz
    . 2006. The immunoregulatory role of IDO-producing human dendritic cells revisited. Trends Immunol. 27: 68–73.
    OpenUrlCrossRefPubMed
  45. ↵
    1. Löb S.,
    2. S. Ebner,
    3. S. Wagner,
    4. J. Weinreich,
    5. R. Schäfer,
    6. A. Königsrainer
    . 2007. Are indoleamine-2,3-dioxygenase producing human dendritic cells a tool for suppression of allogeneic T-cell responses? Transplantation 83: 468–473.
    OpenUrlCrossRefPubMed
  46. ↵
    1. Graca L.,
    2. S. P. Cobbold,
    3. H. Waldmann
    . 2002. Identification of regulatory T cells in tolerated allografts. J. Exp. Med. 195: 1641–1646.
    OpenUrlAbstract/FREE Full Text
  47. ↵
    1. Yang H.,
    2. R. Ding,
    3. V. K. Sharma,
    4. F. S. Hilaire,
    5. M. Lagman,
    6. B. Li,
    7. D. A. Thomas,
    8. X. Luo,
    9. P. Song,
    10. C. Stauffer,
    11. et al
    . 2007. Hyperexpression of Foxp3 and IDO during acute rejection of islet allografts. Transplantation 83: 1643–1647.
    OpenUrlCrossRefPubMed
  48. ↵
    1. Zheng X. X.,
    2. A. Sánchez-Fueyo,
    3. M. Sho,
    4. C. Domenig,
    5. M. H. Sayegh,
    6. T. B. Strom
    . 2003. Favorably tipping the balance between cytopathic and regulatory T cells to create transplantation tolerance. Immunity 19: 503–514.
    OpenUrlCrossRefPubMed
  49. ↵
    1. Cobbold S. P.,
    2. E. Adams,
    3. K. F. Nolan,
    4. F. S. Regateiro,
    5. H. Waldmann
    . 2010. Connecting the mechanisms of T-cell regulation: dendritic cells as the missing link. Immunol. Rev. 236: 203–218.
    OpenUrlCrossRefPubMed
  50. ↵
    1. Fallarino F.,
    2. U. Grohmann,
    3. S. You,
    4. B. C. McGrath,
    5. D. R. Cavener,
    6. C. Vacca,
    7. C. Orabona,
    8. R. Bianchi,
    9. M. L. Belladonna,
    10. C. Volpi,
    11. et al
    . 2006. 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.
    OpenUrlAbstract/FREE Full Text
  51. ↵
    1. Chung D. J.,
    2. M. Rossi,
    3. E. Romano,
    4. J. Ghith,
    5. J. Yuan,
    6. D. H. Munn,
    7. J. W. Young
    . 2009. Indoleamine 2,3-dioxygenase-expressing mature human monocyte-derived dendritic cells expand potent autologous regulatory T cells. Blood 114: 555–563.
    OpenUrlAbstract/FREE Full Text
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