Specific subsets of murine dendritic cells acquire potent T cell regulatory functions following CTLA4-mediated induction of indoleamine 2,3 dioxygenase

Andrew L. Mellor, Phillip Chandler, Babak Baban, Anna M. Hansen, Brendan Marshall, Jeanene Pihkala, Herman Waldmann2, Stephen Cobbold2, Elizabeth Adams2 and David H. Munn1

Department of Medicine and 1 Department of Pediatrics, Program in Molecular Immunology, Institute of Molecular Medicine and Genetics, Medical College of Georgia, 1120, 15th Street, Augusta, GA 30912-2600, USA 2 Therapeutic Immunology Group, Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford, OX1 3RE, UK

Correspondence to: A. L. Mellor; E-mail: amellor{at}mcg.edu


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Murine dendritic cells (DCs) expressing indoleamine 2,3 dioxygenase (IDO) catabolize tryptophan and can suppress T cell responses elicited in vivo. Here, we identify specific subsets of splenic (CD11c+) dendritic cells competent to mediate IDO-dependent T cell suppression following CTLA4-mediated ligation of B7 molecules. IDO-competent DC subsets acquired potent and dominant T cell suppressive properties as a consequence of IDO up-regulation, as they blocked the ability of T cells to respond to other stimulatory DCs in the same cultures. Soluble CTLA4 (CTLA4-Ig) and cloned CTLA4+ regulatory T cells (Tr1D1) up-regulated IDO selectively in DC subsets co-expressing B220 or CD8{alpha}. The ability of Tr1D1 T cells to suppress CD8+ T cell responses was completely dependent on their ability to induce tryptophan catabolism in DCs. Selective IDO up-regulation in DCs did not inhibit T cell activation, but prevented T cell clonal expansion due to rapid death of activated T cells. T cell responses were restored by genetic or pharmacologic inhibition of IDO enzyme activity, or by adding excess tryptophan. DCs from interferon {gamma} (IFN{gamma})-receptor-deficient mice were effective in promoting IDO-dependent T cell suppression following CTLA4-Ig exposure in vivo, indicating that IFN{gamma} signaling was not necessary for IDO up-regulation in this model. These findings suggest that IDO-competent DCs provide a regulatory bridge, mediated by CTLA4-B7 engagement, between certain regulatory T cell subsets and naive responder T cells.

Keywords: dendritic cells, IDO, mice, suppression, T cells


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Dendritic cells (DCs) are key components of afferent processes that lead to effective antigen-specific immune responses following tissue inflammation (1,2). In certain circumstances, antigenic challenge promotes suppressive, or tolerant, rather than immune outcomes. Several reports document experimental conditions in which murine DCs suppress immunity, or promote tolerance by inhibiting T cell responses to tissue auto-antigens and allo-antigens (38). Moreover, tissue inflammation at mucosal surfaces, such as the gastro-intestinal tract and the maternal–fetal interface, is associated with tolerant, rather than immune responses to foreign antigens (9,10). However, the underlying mechanisms employed by DCs to suppress expansion and differentiation of effector T cells and to promote T cell tolerance remain elusive. One contributing factor is the maturation status of DCs at the time of antigen presentation to T cells, which is a key determinant of immune outcomes in some experimental systems. Additionally, some DCs may deploy specific molecular mechanisms allowing them to limit T cell proliferation and/or modify T cell differentiation independently of their maturation status as APCs.

Cells expressing the enzyme indoleamine 2,3 dioxygenase (IDO), which degrades the essential amino acid tryptophan, may deploy this mechanism to suppress T cell proliferation (11). In support of this notion we, and others, have shown that myeloid APCs expressing IDO inhibit T cell responses in vivo (12,13). Experiments with human macrophages and DCs expressing IDO revealed that IDO activity in APCs did not inhibit their ability to activate primary human T cells, but blocked T cell proliferation in vitro (1417). In mice, CD8{alpha}+ DCs preferentially suppressed DTH responses (18) and expressed functional IDO in response to interferon {gamma} (IFN{gamma}) treatment (19). Alternative indirect methods, including transfection of murine tumor cell lines and transgenesis (20), confirmed the inhibitory effects of IDO activity on T cell responses. However, splenic APCs and fractionated CD8{alpha}+ DCs were potent stimulators of primary T cell responses, and responses were not enhanced in the presence of IDO inhibitors (our unpublished data). Hence, in contrast to studies with IDO+ human DCs, IDO-dependent T cell suppression has not been demonstrated directly in studies using physiologic DCs to stimulate primary T cell responses. Consequently, it has not been possible to define specific subsets of murine DCs responsible for IDO-dependent suppression.

Here, we address this deficiency by exploiting recent observations that IDO expression and tryptophan catabolizing activity is induced by exposing murine CD11c+ splenic DCs to CTLA4-Ig, or by culturing them with T cells expressing surface CTLA4, which ligates B7 and induces IDO expression by ‘reverse-signaling’ pathways in DCs (2123). In a previous report (24), we showed that CTLA4-Ig-induced IDO expression completely blocked potent in vivo allogeneic T cell responses and that CTLA4-Ig-induced IDO expression was selective for DC subsets expressing B220, CD8{alpha} and the NK marker DX5, though we did not assess functional properties of these subsets in vitro. In this report, we identify a specific subset of splenic DCs competent to express IDO following B7 ligation that suppress clonal expansion of naive T cells in vitro. We show that IDO-competent DCs are conditioned to lose their T cell stimulatory functions and to acquire potent regulatory functions via CTLA4-mediated IDO up-regulation.


    Methods
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Mice
CBA, C57BL6/J, F1[CBAxB6], BM3 (H2Kb-allospecific, CD8+) and A1.M (H2Ek-restricted, H-Y-specific, CD4+) TCR Tg mice (24,25) were bred in a specific pathogen-free facility at the Medical College of Georgia. IDO-KO mice were generated as described previously (24,26). OVA-specific OT-I and OT-II TCR Tg mice (27,28) were the generous gift of Dr P. Koni (MCG). IFN{gamma}R-KO and 129/SvJ mice were the generous gift of Dr D. Moskofidis (MCG). All procedures were carried out with the approval of the Institutional Animal Care and Use Committee (IACUC).

Peptides
Peptides were prepared and HPLC purified by the MCG Molecular Biology Core Facility using standard methods. The following sequences were used: HYEk, REEALHQFRSGRKPI for A1.M T cells (29) and SIINFEKL for OT-I T cells (27). In experiments using OT-II mice, ovalbumin (albumin chicken egg, grade III, A5378; Sigma, St Louis, MO) or cognate OT-II peptide, AAHAEINEA (28) was used.

CTLA4-Ig
Cytolytic (native) CTLA4-IgG2a was purchased from Sigma (C4483); a mutated (non-cytolytic) CTLA4-IgG2a isotype (C4358) was used as a control in some experiments. These reagents have comparable effects on suppressing proliferation of polyclonal murine T cells via co-stimulatory blockade, according to supplier specifications. In some experiments, cytolytic CTLA4-IgG2a (MF110A4) was purchased from Chimerigen (Allston, MA). Mice received 100 µg CTLA4-Ig (i/p). Isotype-matched purified murine IgG2a was purchased from BD Biosciences (553453, San Diego, CA) and used as a control in some experiments.

Mixed lymphocyte reactions (MLRs)
MLRs were performed essentially as described previously (30). Combinations of responders and stimulators were set up in triplicate wells in a total of 200 µl/well RPMI 1640 medium (15-041-CV, Cellgro, Herndon, VA) supplemented with 10% fetal bovine serum (FBS, Sigma), penicillin (100 i.u./ml) and 100 mg/ml streptomycin (Cellgro), 2 mM/ml L-glutamine (Cellgro) and 5 x 10–5M ß2-mercaptoethanol in 96-well round bottomed plates (Falcon, Bedford, MA). Responder T cells were enriched using nylon wool and used at 1 x 105 cells/well. Stimulators were used at 5 x 105/well and were irradiated in some experiments (30 Gy, Nordian Gammacell Irradiator). Plates were incubated for a total of 72 h at 37°C in a humidified 5% CO2 atmosphere. All wells were pulsed with 0.5 µCi 3H in 40 µl RPMI 1640 for the last 6 h of the incubation period. Thymidine incorporation was measured using the BetaPlate system (Wallac). In experiments shown in Fig. 5, nylon wool enriched BM3 T cells were stained with CFSE (2 µM, 20 min, 37°C) and used in MLRs as described above.



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Fig. 5. IDO+ APCs reduce T cell viability following activation. F1[CBAxB6] (IDO-WT) mice were treated with PBS (triangles) or CTLA4-Ig (circles, squares). CBA mice were treated with CTLA4-Ig as controls. Twenty-four hours later, splenocytes were placed in culture with CFSE-stained BM3 T cells with no additions (squares), 1mT (circles, 100 µM) or excess tryptophan (5xT, 122 µM), as indicated. Cells were harvested after 24–48 h in culture, stained with anti-CD8, anti-CD25 mAbs and subjected to flow cytometric analyses. (A and B) Total numbers of live (FSC, SSC gated) T cells (CFSE+) and stimulator cells (CFSE–), respectively, between 24–48 h in cultures. Two-color dot plots (top row) show CFSE and CD8 staining profiles for live (gated) cells after 48 h in culture. Histograms show CFSE (center row) and CD25 (lower row) staining profiles for live BM3 T cells (CFSE+, CD8+) at this time point. Percent divided (CFSElow, CD8+) relative to undivided (CFSEhigh, CD8+) BM3 T cells are indicated in CFSE histograms. Markers in CD25 histograms were set to exclude naive (CD25–) T cells in cultures containing stimulators from CBA mice. Cultures were analyzed in triplicate and data shown are representative of three independent experiments.

 
1-methyl-tryptophan and tryptophan
1 methyl-D-tryptophan (Aldrich, Milwaukee, WI; F.W. 218.6 m.p. 250°C) was added to a final concentration of 100 µM. L-tryptophan (Sigma, L-a-amino-3-indole-propionic acid; FW204.2) was added as a supplement to final concentrations of 122.5 µM or 245 µM, equivalent to x5 or x10 the original concentration (24.5 µM) in RPMI 1640.

Tr1D1 regulatory T cells
The male (H-Y) specific H-2Ek restricted CD4+ T regulatory clone (Tr1D1) was originally generated from naive A1(M)xRag-1-KO mice (31). Clones were maintained on a 14 day re-stimulation cycle using 2 x 105 clone with 5 x 105 irradiated CBA male spleen cells in 10% FBS/RPMI supplemented with IL-2 (20 U/ml) and IL-4 (20 U/ml). In MLR assays, non-irradiated splenocytes (7.5 x 104) from F1[CBAxB6] mice were used to stimulate nylon-wool enriched BM3 T cells (2.5 x 104) in the presence or absence of Tr1D1 T cells (2.5 x 104, harvested 10 days after re-stimulation) in 96-well round bottom plates in triplicate wells. Anti-CTLA4 mAb (clone 9H10, Cat. No. 16-1521-85; eBiosciences, San Diego, CA) was added at 100 µg/ml to block CTLA4-B7 interactions in some cultures. Tritiated [3H]thymidine (0.5 µCi/well) was added for the final 6 h of a 72 h incubation. Plates were harvested and thymidine incorporation measured using the BetaPlate system. For cytospin preparations, 5 x 105 Tr1D1 and 5 x 104 DCs were co-cultured in 200 µl of 10% FBS/RPMI in 96-well round bottom plates for 48 h prior to harvest.

Isolation of splenic DC subsets
Spleens were harvested into 1% FBS/HBSS. One milliliter of collagenase IV (100 CD units/ml in 1% FBS/HBSS; CLS-4; Worthington, Lakewood, NJ) was injected into three areas of each spleen. Injected spleens were then placed in collagenase IV (1 ml/spleen of 400 CD units/ml in 1% FBS/HBSS). After incubation (37°C, 30 min) spleens were made into a single cell suspension, centrifuged (250 g, 1300 r.p.m., 5 min), and erythrocytes lysed (3 min) in 3 ml of ACK lysing buffer (10-548E, BioWhittaker, Walkersville, MD). Splenocytes were washed twice (10 mM EDTA in Ca/Mg-free PBS) before fractionation (MACS) or sorting (Mo-Flo) as described below.

AutoMACS fractionation
Cell pellets were resuspended in running buffer (1% BSA, or 2% FCS in 1 mM EDTA in Ca/Mg-free PBS), and anti-murine CD11c microbeads (130-052-001, Miltenyi, Auburn, CA) were added (50 µl/ml). Following incubation (30 min on ice) in the dark, cells were washed twice and CD11c+ cells were selected using the AutoMACS system. Typically, CD11c+ cells isolated by this procedure were 80–85% pure, while CD11c– cells were >99% pure.

Preparative flow cytometry
For isolation of CD11c+B220+ and CD11c+CD8{alpha}+ splenic DC subsets, splenocyte suspensions were incubated with a cocktail of either PE-CD11c (557401, Pharmingen, San Diego, CA) and FITC–B220 (553088, Pharmingen), or PE–CD11c and FITC–CD8{alpha} (553031, Pharmingen) for 20 min (4°C). Preparative cell sorting was performed using a Mo-Flo 4-way flow cytometer to select cells of interest using DakoCytomation SummitTM software (DakoCytomation, Fort Collins, CO). CD11c+ cell fractions were selected for high purity (95–99%), which was achieved by setting sorting gates to collect cells unambiguously stained by CD11c mAb. This procedure sacrificed some DCs with lower CD11c staining profiles (CD11clow), but avoided contamination with macrophages and other cell types whose autofluorescence overlapped the CD11clow region. As shown in the Results section, essentially all IDO-dependent T cell suppressive activity segregated with the unambiguous CD11c+ sorted cells, so it was not necessary to include ambiguous CD11clow populations for the purposes of this study. Sorting gates for B220 and CD8{alpha} staining were set between distinct populations of stained/unstained cells. All sorted DC subsets exhibited comparable light scatter properties (FSChigh, SSChigh) characteristic of large mononuclear cells.

Anti-IDO antibody
Polyclonal rabbit anti-murine IDO antibody was prepared by a commercial supplier (Biosource International, Hopkinton, MA). Antisera were raised against two synthetic peptides (KPTDGDKSEEPSNVESRGC, CSAVERQDLKALEKALHD) following conjugation to ovalbumin. Antisera were affinity purified over the first peptide and screened for reactivity by ELISA.

Immunohistochemistry
Tissue sections (5 µm) were prepared from formalin-fixed paraffin-embedded tissues. Following deparaffinization, sections were washed for 10 min in distilled water. Cytospin preparations of ~20 000 sorted cells/sample chamber were centrifuged (50 g, 700 r.p.m., 5 min) air-dried, fixed in 10% formalin, and washed twice in PBS. All subsequent procedures were carried out at room temperature. Endogenous peroxidase activity was blocked with hydrogen peroxide (1:10 w/PBS, 10 min). Tissue sections were also treated with proteinase K (S3020, DAKO) for 10 min. After two washes in PBS, all preparations were treated with Universal Blocking Reagent (HK085-5K, BioGenex, San Ramon, CA; 1:10 in distilled water), rinsed in PBS, and incubated with IDO antibody (1:100 in PBS; 1 h for cytospins, 2 h for tissue sections). After two washes in PBS, preparations were treated with biotinylated goat anti-rabbit Ig (HK336-9R, BioGenex). After a 5 min wash in PBS, slides were incubated for 20 min in peroxidase-conjugated Streptavidin (HK330-9K, BioGenex). IDO-expressing cells were visualized using 3-amino-9-ethylcarbazole chromogen (HK121-5K Liquid AEC, BioGenex) for >30 s, <10 min. Preparations were counterstained with hematoxylin (7221, Richard-Allan Scientific, Kalamazoo, MI) and mounted in Faramount (S3025, DAKO). IDO antibody pre-incubated with neutralizing peptide (1.2 µg antibody:10 µg peptide) was used as the specificity control.

Analytical flow cytometry
Three color flow cytometric analyses were performed on a Becton-Dickenson FACSCaliburTM using CellQuestTM software. CFSE-stained, nylon wool enriched BM3 T cells were identified by staining with APC-conjugated anti-CD8 mAb (clone 53-6.7) after gating to exclude dead cells and debris using forward and side scatter plots. T cell activation was measured by staining with PE-conjugated anti-CD25 mAb (clone 3C7). Absolute numbers of T cells were calculated after calibrating flow rates using Flow-CountTM fluorospheres (Beckman Coulter). All conjugated mAbs were purchased from BD Pharmingen.


    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
CTLA4-Ig conditions splenic APCs to suppress T cell proliferation by inducing IDO expression
To assess the consequences of IDO up-regulation on T cell stimulatory functions of splenic APCs we exposed F1[CBAxB6] IDO-WT and IDO-KO mice to CTLA4-Ig for 24 h (24), and then used splenocytes from treated mice as stimulators in MLRs (Fig. 1). Responder T cells originated from four different lines of TCR Tg mice, BM3 (H-2Kb-specific, CD8+), A1 (male H-Y antigen specific, CD4+), OT-I and OT-II (ovalbumin-specific, CD8+ and CD4+) T cells. In every case, splenocytes from CTLA4-Ig-treated IDO-WT mice were poor stimulators of T cell proliferation, generating responses that were 70–95% less than responses stimulated by splenocytes from untreated IDO-WT mice. However, exposure to CTLA4-Ig in vivo had no significant effects on T cell proliferative responses stimulated by splenocytes from IDO-KO mice. Thus, the immunomodulatory properties of CTLA4-Ig were almost completely IDO-dependent in this experimental system. IDO-WT mice exposed to isotype-matched IgG2a antibody yielded splenic APCs that stimulated T cell proliferation as effectively as APCs from untreated mice (data not shown), indicating that IgG2a binding to Fc receptors or complement factors did not explain the effects of CTLA4-Ig exposure in vivo on T cell stimulatory properties of APCs in vitro.



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Fig. 1. CTLA4-Ig-mediated T cell suppression is IDO-dependent. IDO-WT or IDO-KO mice were injected (i/p) with PBS (–) or 100 µg CTLA4-Ig (+). After 24 h, mice were sacrificed, and irradiated (3000 Rads) splenocytes were used as stimulators in co-cultures with nylon-wool enriched responder T cells from BM3, A1, OT-I or OT-II TCR transgenic mice, as indicated. T cell proliferation was assessed by thymidine incorporation after 72 h. Stimulators were isolated from F1[CBAxB6] (H-2b/k) mice for BM3, OT-I, OT-II responder T cells or from female CBA (H-2k) mice for A1 responder T cells. Stimulators were pulsed with synthetic peptides (REEALHQFRSGRKPI, A1; SIINFEKL, OT-I) or ovalbumin (OT-II). Percent reduction in thymidine incorporation stimulated by APCs from CTLA4-Ig-treated IDO-WT mice, relative to untreated controls is shown for each responder T cell. Results were calculated as the mean of triplicate cultures (+1 SD) and were replicated at least twice.

 
IDO-dependent T cell suppression is mediated by specific DC subsets
Next, we fractionated splenic APC subsets from CTLA4-Ig-treated F1[CBAxB6] mice, and assessed their ability to promote BM3 T cell proliferation (Fig. 2). We selected BM3 T cells as responders because most cells expressing H-2Kb stimulate BM3 T cell proliferation (20). As before, T cell responses to unfractionated (total) splenic APCs from IDO-WT mice were reduced significantly by CTLA4-Ig exposure in vivo (Fig. 2A, black bars), but this treatment had no significant effect on responses elicited by APCs from IDO-KO mice (data not shown). Addition of IDO inhibitor (1mT) to MLRs restored T cell proliferation to control levels, relative to responses elicited in cultures containing APCs from untreated mice (Fig. 2A, white bars). This procedure revealed that a substantial component of underlying T cell stimulatory properties of APCs was effectively concealed by IDO enzyme activity. CTLA4-Ig exposure in vivo had no effects on T cell stimulatory properties of splenic APCs that were not IDO-dependent in this system.



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Fig. 2. DC subsets expressing B220 and CD8{alpha} mediate IDO-dependent T cell suppression. F1[CBAxB6] IDO-WT mice were injected with PBS (–) or 100 µg CTLA4-Ig (+). After 24 h, splenocytes were stained with mAb cocktails and fractionated using MACS, or sorted by (Mo-Flo) flow cytometry as detailed in Methods. The CD11c staining profile at top left shows how Mo-Flo sorting gates were set to isolate highly purified CD11c+ cells (95–99% CD11c+). Two-color staining profiles show how sorting gates were set to separate CD11c+ cells stained or unstained by B220 or CD8{alpha} mAbs (see Methods). Relative proportions of the four sorted DC subsets (expressed as % of total sorted CD11c+ cells) are indicated on two-color plots. Fractionated, or sorted splenocytes (non-irradiated) were cultured with nylon-wool enriched H-2Kb-specific BM3 T cells and T cell proliferation was assessed by thymidine incorporation after 72 h (black bars). IDO inhibitor, 1mT was added to parallel cultures (white bars). (A) MLRs using unfractionated (total) and MACS-enriched CD11c+ and CD11c– fractions. (B and C) MLRs using Mo-Flo sorted DC subsets, and total CD11c+ (B), or total splenocytes (C) as indicated. Arrows indicate percent reduction in T cell proliferative responses due to IDO-mediated inhibition, relative to responses elicited in cultures containing IDO inhibitor. Cultures were analyzed in triplicate (+1 SD), and each sort was conducted on at least two separate occasions.

 
Using MACS cell fractionation to enrich for splenocytes expressing CD11c+ we found that CTLA4-Ig treatment in vivo significantly reduced the ability of CD11c+ cell fractions (DCs) to stimulate in vitro T cell proliferation (Fig. 2A). Low T cell proliferation rates were not caused by over-stimulation of T cell responses by purified CD11c+ cells because cultures remained quiescent (based on daily visual inspection), and because addition of 1mT restored T cell proliferation rates to control levels. In contrast, in vivo CTLA4-Ig treatment had no significant effects on the ability of remaining CD11c– splenocytes (non-DCs) to stimulate T cell proliferation, indicating that these cells did not mediate IDO-dependent T cell suppression. T cell stimulatory properties of sorted CD11c+ and CD11c– cells from IDO-KO mice were not affected by in vivo CTLA4-Ig treatment (data not shown). CD11c– splenocytes, which included macrophages and lymphocytes, were also excellent APCs for BM3 responder T cells. However, the underlying T cell stimulatory capacity of splenic CD11c– APCs from CTLA4-Ig-treated mice was not evident until they were separated from CD11c+ DCs, indicating that IDO-dependent suppression was dominant over APC stimulatory functions in this system. BM3 responsiveness to CD11c– splenocytes was robust, reflecting the promiscuity of these CD8+ T cells, which proliferate vigorously to most cell types expressing H-2Kb, including non-APC cells and cell lines (20).

To identify DC subsets that mediated IDO-dependent T cell suppression we used flow cytometry (Mo-Flo) to sort specific DC subsets (see Methods), based on expression of CD11c and B220 (Fig. 2B), or CD11c and CD8{alpha} (Fig. 2C). Using this approach, we found that IDO-dependent suppression of BM3 T cell proliferation was a unique characteristic of specific subsets of sorted splenic CD11c+ DCs co-expressing either B220 or CD8{alpha}. In contrast, CTLA4-Ig exposure in vivo had no significant IDO-dependent effects on the T cell stimulatory properties of CD11c+ DCs not expressing these markers. Immunohistochemical analyses of IDO expression in sorted DC subsets revealed that almost all CD11c+B220+CD8{alpha}+ cells were IDO+, and that this minor subset of DCs (~10–14% of sorted CD11c+ cells) accounted for most of the IDO+ cells isolated from spleens of CTLA4-Ig-treated mice (24) (data not shown). In spite of the low numbers of IDO+ DCs, the underlying T cell stimulatory properties of other DCs (CD11c+B220–, CD11c+CD8{alpha}–) were censored by their presence in cultures.

Cloned CTLA4+ regulatory T cells promote IDO-dependent T cell suppression
To assess if cell surface CTLA4 expression induced IDO up-regulation selectively in IDO-competent DCs, we cultured sorted CD11c+ DCs with cloned CD4+CD25+ T cells (Tr1D1) expressing high levels of surface CTLA4 (31). After co-culture for 48 h, ~70% of Mo-Flo sorted CD11c+B220+ and CD11c+CD8{alpha}+ DCs expressed IDO, whereas <10% were IDO+ when DCs were cultured alone (Fig. 3). Tr1D1 cells also increased the proportion of DCs expressing IDO in sorted B220– and CD8{alpha}– DC subsets, though increases were statistically significant only in B220+ and CD8{alpha}+ subsets. In vitro exposure to IFN{gamma} also increased the proportion of DCs expressing IDO and increases were most marked in B220+ and CD8{alpha}+ subsets (19) (data not shown). However, tryptophan catabolism was detected in CD8{alpha}+, but not CD8{alpha}– DCs (19), suggesting that IDO protein expression is not sufficient for enzyme activity in CD8{alpha}– DCs. These data demonstrate that Tr1D1 T cells, like IFN{gamma} and CTLA4-Ig preferentially induced IDO expression in DC subsets expressing B220 or CD8{alpha}, which are competent to express functional IDO and mediate IDO-dependent T cell suppression.



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Fig. 3. CTLA4+ cells preferentially induce IDO expression in B220+ and CD8{alpha}+ DCs. Cloned CTLA4+ Tr1D1 cells were cultured with Mo-Flo sorted CD11c+B220+, CD11c+B220–, or CD11c+CD8{alpha}+, CD11c+CD8{alpha}– DCs from F1[CBAxB6] IDO-WT mice. After 48 h, cultures were collected by cytospin and stained to detect IDO+ cells. Cells with DC morphology were scored as IDO+ or IDO– by inspection of 10 randomly selected image fields, and results were plotted as bar graphs showing the proportion of IDO+/total DCs.

 
Cloned Tr1D1 T cells possess potent T cell regulatory functions that inhibit Th1 and Th2-mediated rejection of skin allografts in mice (31). To test the hypothesis that regulatory properties of Tr1D1 cells actually depend on their ability to up-regulate IDO in APCs, we added Tr1D1 T cells to cultures containing responder BM3 T cells mixed with MACS enriched CD11c+ APCs from F1[CBAxB6] IDO-WT, or IDO-KO mice (Fig. 4A). When APCs originated from IDO-WT mice, Tr1D1 T cells efficiently suppressed BM3 T cell proliferation (80% suppression). When CD11c+ APCs originated from IDO-KO mice, the effect of adding Tr1D1 cells was diametrically opposed, with significant enhancement of BM3 T cell proliferation (5x control levels). Thus, the ability of DCs to express IDO was critical for Tr1D1-mediated suppression of BM3 T cell proliferation. Consistent with an obligatory role for IDO-competent DCs in Tr1D1-mediated suppression, addition of IDO inhibitor (vertical striped bars) or excess tryptophan (horizontal striped bars) to cultures containing Tr1D1 cells restored BM3 T cell proliferation to levels significantly in excess of responses elicited in cultures containing DCs from IDO-WT mice alone (Fig. 4B). Moreover, significantly enhanced BM3 proliferation was observed when blocking anti-CTLA4 mAb was added to MLRs containing Tr1D1 cells and IDO-WT APCs (Fig. 4B, diagonally striped bars), indicating that CTLA4-B7 interactions were essential for Tr1D1-mediated regulatory functions. However, Tr1D1 cells had little effect on BM3 T cell proliferation stimulated by CD11c– APCs from IDO-WT mice (Fig. 4C). These data show that Tr1D1 cells suppressed BM3 T cell proliferation by up-regulating IDO expression in IDO-competent DCs. Indeed, when APCs could not deplete tryptophan, Tr1D1 cells not only lost their regulatory properties, but gained T cell stimulatory functions. Presumably, enhanced T cell stimulation in the absence of functional IDO expression and presence of Tr1D1 cells was due to more efficient DC maturation, a response that is normally masked by the potent regulatory effects of induced IDO activity. The identity of these maturation mechanisms is not known, but is not due to B7 ligation since blockade of CTLA4-B7 interactions by anti-CTLA4 mAb significantly enhanced BM3 responses. These findings with Tr1D1-mediated IDO-dependent suppression of BM3 T cell proliferation were consistent with our studies on CTLA4-Ig-mediated IDO-dependent T cell suppression.



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Fig. 4. Tr1D1 cells suppress T cell responses by conditioning APCs to express IDO. BM3 responder T cells and APCs from F1[CBAxB6] male mice were cultured with or without (black bars) Tr1D1 T cells for 72 h, and T cell proliferation was assessed by measuring thymidine incorporation. (A) Effect of Tr1D1 cells on T cell responses in the presence of MACS-enriched CD11c+ (80–85% pure) DCs from IDO-WT and IDO-KO mice. (B) Effects of IDO inhibitor, 1mT (vertical stripes), excess tryptophan (10xT, horizontal stripes) and addition of anti-CTLA4 mAb (100 µg/ml, diagonal stripes) on Tr1D1-mediated suppression of T cell responses primed by CD11c+ DCs from IDO-WT mice. (C) As above, except that CD11c– (>99% pure) cells were used as APCs. Experiments were executed three times in triplicate (+1 SD).

 
Tryptophan depletion by IDO+ DCs inhibits T cell proliferation and promotes cell death
To discover the fate of T cells cultured with IDO+ DCs, we incubated CFSE-labeled BM3 T cells with splenocytes from untreated and CTLA4-Ig-treated F1[CBAxB6] mice and assessed T cell numbers, their division status and CD25 expression by flow cytometry over time (Fig. 5). Just before the first cell division (at ~24 h) numbers of live (gated by light scatter properties) responder T cells (CFSE+) and stimulator (CFSE–) cells were comparable in all cultures (Fig. 5A and B; and data not shown). After 40 h, numbers of live CFSE+ T cells in cultures containing IDO+ APCs (squares) were reduced dramatically to <1% of live cell numbers in cultures containing APCs from untreated mice (triangles), or from IDO-KO mice exposed to CTLA4-Ig (Fig. 5A; and data not shown). The viability of live stimulator (CFSE–) cells was also reduced, but to a significantly lesser degree (Fig. 5B), indicating that responder BM3 T cells died rapidly and preferentially in the presence of IDO+ DCs. After 48 h, T cell numbers were still relatively low in cultures containing IDO+ DCs, but numbers appeared to be recovering; it is not clear if delayed recovery was due to clonal expansion of viable cells, or due to CFSE recycling. Addition of 1mT (circles) restored BM3 T cell clonal expansion in cultures containing IDO+ APCs to control levels (Fig. 5A).

After 48 h of culture, FACS staining profiles for CFSE and CD25 revealed that the few surviving BM3 T cells had divided in cultures containing IDO+ APCs, and that these T cells all expressed CD25. Thus, in the presence of IDO+ APCs the few remaining viable BM3 T cells showed evidence of activation, but clonal expansion was severely limited, either through slower division rates, lower T cell viability, or a combination of both processes. Since 1mT, and excess tryptophan, restored T cell responses the major cause of reduced T cell proliferation and viability was limited access to tryptophan, and not toxic metabolite production by IDO+ DCs. As expected, splenic APCs from CTLA4-Ig-treated IDO-KO mice stimulated potent T cell proliferative responses, comparable with responses provoked by APCs from untreated IDO-WT mice (data not shown). Thus, CTLA4-Ig-mediated IDO up-regulation in DC subsets caused rapid and preferential death of activated T cells due to tryptophan depletion.

IDO-mediated T cell suppression does not require IFN{gamma} signals
IFN{gamma} is a potent inducer of IDO gene transcription (32). To test if CTLA4-Ig induced IDO expression was IFN{gamma}-dependent, we evaluated IDO-dependent T cell suppression in IFN{gamma}-receptor deficient (IFN{gamma}R-KO) mice, in which APCs cannot receive signals from IFN{gamma}. Immunohistochemical analyses revealed that CTLA4-Ig exposure induced comparable numbers of splenocytes (~3%) to express IDO in IFN{gamma}R-KO and WT (129/SvJ) mice (Fig. 6A and B). In contrast, few (<0.1%) splenocytes expressed IDO in mice exposed to a mutated, non-cytolytic isotype of CTLA4-Ig with a modified IgG2a Fc domain (Fig. 6C). As expected (24), most IDO+ cells were located in splenic red pulp areas after CTLA4-Ig treatment. Thus, inability to receive signals from IFN{gamma} had no effect on CTLA4-Ig-induced IDO up-regulation in IDO-competent splenocytes.



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Fig. 6. IDO-dependent T cell suppression is IFN{gamma}-independent. F1[CBAxB6] (A) and IFN{gamma}R-KO (B) mice were exposed to CTLA4-Ig as before. F1[CBAxB6] mice were also exposed to a mutant (non-cytolytic) CTLA4-Ig isotype, as described in Methods (Fig. 6C). After 24 h, spleens were prepared for immunohistochemical analysis to detect IDO+ cells. Original magnifications x200. (D and E) IFN{gamma}R-KO and 129/SvJ mice were treated with PBS (–), or CTLA4-Ig (+) and T cell stimulatory properties of splenic APCs were assessed as before. Cultures contained no additions (black bars), 1mT (white bars), or excess (5xT) tryptophan (striped bars). Arrows indicate percent CTLA4-Ig-mediated T cell suppression, relative to untreated controls.

 
To test if IDO up-regulation in IFN{gamma}R-KO mice was functionally significant, we assessed T cell proliferation in MLRs containing APCs from CTLA4-Ig-treated IFN{gamma}R-KO mice (Fig. 6D and E). CTLA4-Ig treatment in vivo had comparable inhibitory effects on T cell responses elicited in vitro when APCs originated from 129/SvJ (IFN{gamma}R-WT) and IFN{gamma}R-KO mice. In each case, ~80% of T cell stimulatory potential was censored by IDO activity. As expected, robust T cell responses were restored in the presence of IDO inhibitor or excess tryptophan, confirming that IDO+ APCs suppressed T cell proliferation by catabolizing tryptophan. These data revealed that in vivo exposure to CTLA4-Ig up-regulated IDO via IFN{gamma}-independent pathways, and converted IDO-competent APCs into regulatory APCs that actively suppressed T cell proliferation.


    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
In this report, we identify relatively minor subsets of splenic DCs that were induced to express IDO, and acquired potent and dominant IDO-dependent regulatory APC functions following CTLA4-dependent conditioning. Moreover, the T cell regulatory properties of cloned Tr1D1 cells were completely dependent on their ability to induce IDO in APCs in our experimental system. In the absence of signals from CTLA4, or in APCs lacking a functional IDO gene, splenic DCs did not express IDO, and were instead potent stimulatory APCs. Thus, APC conditioning to induce IDO expression in minor subsets of DCs was a critical factor influencing T cell proliferative responses elicited in vitro.

The hypothesis that DCs expressing IDO suppress T cell proliferation is supported by evidence from several sources (12,13). Indirect evidence supporting this hypothesis comes from pharmacologic blockade of the regulatory properties of murine CD8{alpha}+ DCs in T cell mediated responses elicited in vivo (18,21), and from studies with IDO-transfected murine tumor cell lines used as T cell stimulators and with cloned Th1 T cells used as responders (19,20). Direct demonstration of IDO-dependent suppression of in vitro T cell proliferation has been reported previously for human DCs expressing IDO (1517). However, studies showing that murine DCs expressing IDO suppress primary T cell responses in vitro have not been reported previously. Physiologic DCs isolated directly from mice normally possess potent T cell stimulatory functions, and addition of IDO inhibitor has no effect on T cell responses elicited in vitro (our unpublished data). In this study, we show that some splenic DC subsets (IDO-competent DCs) acquired potent T cell suppressive properties following B7 ligation in vivo or in vitro, which were reversed in the presence of IDO inhibitor, excess tryptophan, or anti-CTLA4 blocking antibody. Hence, T cell regulatory properties of CTLA4-conditioned APCs depended on the ability of IDO-competent DCs to reduce tryptophan concentrations following B7-mediated IDO up-regulation. These data also revealed that tryptophan metabolites produced by IDO+ APCs were not responsible for suppressing proliferation of primary T cells following antigen-specific activation. Previous studies using human T cells, or cloned murine CD4+ T cells as responders, reported that some metabolites produced by cells expressing IDO inhibited T cell proliferation, prompting the suggestion that IDO-dependent inhibition of T cell proliferation was caused by production of toxic metabolites, which enhanced T cell apoptosis (3335). However, in the experimental systems described here, and in previous studies with human macrophages and DCs (14,17), tryptophan depletion was the key biochemical process that inhibited T cell responses, since addition of excess tryptophan fully restored T cell proliferation, even though this manipulation increased metabolite production.

Previously, CTLA4-Ig was shown to induce IDO in isolated splenic DCs by ligating B7 molecules (21). In the current study, we confirmed and extended this finding by identifying specific subsets of DCs that mediate potent and dominant IDO-dependent T cell suppression in vitro following CTLA4-Ig exposure in vivo. We showed that B220 and CD8{alpha} subsets contained IDO-competent DCs, while DCs that did not express these markers did not mediate IDO-dependent T cell suppression, even though some IDO+ DCs were detected in these DC subsets by cytospin analyses. Since IgG2a treatment or CTLA4-Ig treatment did not reduce potent stimulatory functions of APCs prepared from IDO-WT mice or IDO-KO mice, respectively, effector functions in the native IgG2a Fc domain of the CTLA4-Ig reagent used in these studies did not induce the potent regulatory effects of CTLA4-Ig exposure in IDO-WT mice. This precludes complement fixation and antibody-dependent cell-mediated cytolysis as potential IDO-independent explanations for regulatory outcomes observed. However, the native Fc domain was essential for effective induction of IDO expression (Fig. 6C) and IDO-dependent T cell suppression (data not shown). We do not know why the native Fc domain structure is essential for efficient induction of IDO, but binding to Fc-receptors on adjacent cells, or to complement factors may enhance B7-CTLA4-Ig molecular interactions on DCs.

IFN{gamma} signaling was not essential for CTLA4-Ig-mediated IDO up-regulation, or IDO-dependent T cell suppression in our experimental system, implying that B7 ligation induced IDO gene expression via IFN{gamma}-independent signaling pathways in DCs. The IDO gene promoter is responsive to IFN{gamma}-mediated signaling via STAT-1-mediated signaling pathways (11,32). However, previous reports showed that CTLA4-Ig did not induce IDO expression when DCs originated from IFN{gamma}-KO mice, suggesting that recruitment of p38 MAPK and NF-{kappa}B were essential for IFN{gamma}-dependent IDO up-regulation following B7 ligation (21). It is unclear why distinctly different roles for IFN{gamma} in IDO up-regulation emerged from these two studies, but differences in experimental approaches, including mouse strain-specific factors, likely explain these disparities. Alternatively, IDO-competent DCs may develop in IFN{gamma}R-KO mice, but not in IFN{gamma}-KO mice, due to differential compensatory processes. This issue notwithstanding, data presented here revealed that IFN{gamma}-mediated signaling is not obligatory for induction of IDO gene expression in some DC subsets following B7 ligation. This finding implies that B7 ligation may signal STAT-1 activation directly, or indirectly, by inducing expression of alternative ligands that can promote STAT-1 activation, such as type-1 interferons.

CTLA4-mediated conditioning of DC functions due to IDO up-regulation may explain how some regulatory T cells expressing surface CTLA4 suppress T cell responses (22). Support for this model came from a recent study showing that CTLA4-transfected Jurkat T cells and physiologic regulatory T cells enhanced tryptophan catabolism in murine DCs (23). In the current study, we demonstrate that T cell regulatory functions of cloned Tr1D1 cells were abrogated completely if APCs could not catabolize tryptophan. By analogy to a model proposed to explain T helper functions (36), we propose that IDO-competent DCs may function as temporal bridges that can be ‘conditioned’ by regulatory T cells expressing CTLA4 to acquire IDO-dependent regulatory functions as an immediate or later response by the pre-conditioned DC. This mechanism provides a potential link between T cells and DCs with regulatory functions, and a potential explanation for the ability of DCs to take on distinct functions in response to different conditioning signals. It is not clear how representative cloned Tr1D1 cells are of physiologic T cells with regulatory functions (Tregs). Tr1D1 cells resemble Tregs in that they express CD25, surface CTLA4 and GITR, and suppress Th1 and Th2 mediated skin allograft rejection (31). However, they do not express foxP3 or IFN{gamma}, but do secrete IL-10, suggesting that they may more closely resemble a special regulatory or anergic state of a Th2 cell than natural Tregs (37). It may be significant that physiologic Tregs (23) and Tr1D1 cells both induced IDO in DCs via constitutive expression of surface CTLA4. One possibility is that foxP3+ Tregs and foxP3– Tr1 cells may act synergistically in maintaining tolerance to allogeneic transplanted tissues, and that the ability of both regulatory T cell subsets to induce IDO expression in DCs may contribute to tolerant outcomes. This question aside, data reported here imply that the key difference between stimulatory and regulatory CD4+ T cells is constitutive expression of surface CTLA4 molecules that ligate B7 molecules on IDO-competent DCs.

In previous studies (24), we showed that CTLA4-Ig exposure in vivo selectively induced IDO expression in CD11c+ DCs co-expressing B220, CD8{alpha}, or the NK-DC marker DX5 (7). Though the degree of overlap between DC subsets expressing these markers is not known accurately, it is clear that B7 ligation elicits different responses from distinct DC subsets. Mechanisms that explain this dichotomy of response are not known, but one possibility is that competency to express IDO is acquired during DC development, so that IDO-competent DCs and IDO-incompetent DCs segregate within developmentally distinct DC lineages. Though IDO-competent DCs express B220, they display several features that appear to distinguish them from typical splenic plasmacytoid DCs described previously (3841). For example, following CTLA4-Ig treatment in vivo, IDO+ DCs were located in splenic red pulp, expressed relatively high levels of CD11c and co-expressed CD8{alpha}, were relatively large cells based on light scatter properties, and displayed a mature phenotype based on analyses of B7 and MHC expression. In contrast, typical splenic plasmacytoid DCs have been reported to express lower levels of CD11c, accumulate preferentially in lymphoid follicles, express CD8{alpha} only when activated, and generally display immature phenotypes. Previous reports (6,18,38,39,4244) have documented that murine DC subsets co-expressing B220 and/or CD8{alpha} can exhibit regulatory functions. We hypothesize that IDO-competent DCs, which comprise a subset of these cells, may mediate a component of their regulatory functions, providing that they receive CTLA4-B7-mediated signals that induce IDO expression.


    Acknowledgements
 
We are grateful for expert technical assistance by Doris McCool, Erika Thompson and Anita Wylds. We thank Pandelakis Koni for comments on the manuscript. These studies were supported by NIH grants AI44219 and HD41187 (to A.L.M.) and CA103320 (to D.H.M.) and by the generous support from the Carlos and Marguerite Mason Trust.


    Abbreviations
 
1mT   1-methyl-(D,L)-tryptophan
DC   dendritic cell
FBS   fetal bovine serum
IDO   indoleamine 2,3 dioxygenase
IFN{gamma}   interferon {gamma}
IFN{gamma}R   IFN{gamma}-receptor deficient
KO   knockout
MACS   magnetic activated cell sorting
MLR   mixed lymphocyte reaction
Treg   T cell with regulatory functions

    Notes
 
Transmitting editor: E. Simpson

Received 21 April 2004, accepted 6 July 2004.


    References
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 Abstract
 Introduction
 Methods
 Results
 Discussion
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