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-Chain and Induce a Regulatory Phenotype in Naive T Cells1
* Department of Experimental Medicine, University of Perugia, Perugia, Italy;
Institut National de la Santé et de la Recherche Médicale Unité 580-Institut Fédératif de Recherche Necker Enfants-Malades, Hôpital Necker, Paris, France;
Department of Biology, The Pennsylvania State University, University Park, PA 16802; and
Julia McFarlane Diabetes Research Centre, University of Calgary, Calgary, Alberta, Canada
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Abstract |
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-chain in murine CD8+ T cells. TCR down-regulation can be demonstrated in vivo and is associated with an impaired cytotoxic effector function in vitro. The longer-term effects of tryptophan catabolism include the emergence of a regulatory phenotype in naive CD4+CD25– T cells via TGF-
induction of the forkhead transcription factor Foxp3. Such converted cells appear to be CD25+, CD69–, CD45RBlow, CD62L+, CTLA-4+, BTLAlow and GITR+, and are capable of effective control of diabetogenic T cells when transferred in vivo. Thus, both tryptophan starvation and tryptophan catabolites contribute to establishing a regulatory environment affecting CD8+ as well as CD4+ T cell function, and not only is tryptophan catabolism an effector mechanism of tolerance, but it also results in GCN2-dependent generation of autoimmune-preventive regulatory T cells. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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and activation of IDO, including CD4+ T cells (15) and polymorphonuclear leukocytes (16). The wide spectrum of physiopathologic conditions in which IDO appears to be at work suggests that multiple mechanisms are used by this effector system to down-regulate T cell and inflammatory responses. IDO can deplete tryptophan in local tissue microenvironments, and thus prevent T cell proliferation (2), but can also generate immunotoxic kynurenines resulting in T cell apoptosis (1). Neither mechanism alone can explain the diversity (3, 5, 6, 8), durability (4), and Ag specificity (10) of the IDO-mediated suppressive effect. Naive CD4+ T cells and Th2 cells are resistant to apoptosis induced by kynurenines (17), and CD8+ T cells are resistant to activation blockade by tryptophan depletion when exposed to strong antigenic stimuli (15).
Reversible impairment of T cell function through down-regulation of the TCR -chain could be a physiological means of down-regulating exacerbated immune responses (18). This phenomenon has also been observed in several pathologic settings, including chronic infection, neoplasia, and autoimmune disease (19). Tregs are crucial in regulating self-reactive T cells in the periphery and ameliorating established inflammation in experimental models (20, 21, 22). They are usually described as CD4+CD25+CD69– cells that inhibit the activation and proliferation of CD4+CD25– lymphocytes via mechanisms ranging from cell-cell contact to the production of the inhibitory cytokines IL-10 and TGF- (23). In vitro-expanded Ag-specific Tregs can suppress autoimmune diabetes (24).
In a microbial infection model in which exacerbation of the immune response is avoided through the action of Tregs, blockade of IDO results in an impaired generation of Tregs and in overwhelming immunopathology (16). Successful treatment of autoimmune neuroinflammation with a synthetic tryptophan metabolite associated with the development of Treg activity has recently been reported (25). Thus, it is possible that besides generalized cell cycle arrest due to sustained tryptophan deprivation, IDO contributes to fine-tuning immune responsiveness by multiple mechanisms that are coordinate but differentiated for specific T cell subsets. We report that early down-regulation of the TCR -chain in CD8+ cells and longer-term induction of a tolerogenic phenotype in naive CD4+ T cells result from the combined effects of tryptophan starvation and tryptophan metabolites. Both of these effects require the general control non-derepressing 2 (GCN2) protein kinase, which is responsive to starvation for amino acids. The CD8+ T cells display impaired function ex vivo, whereas the CD4+ Tregs are shown to prevent disease in an experimental setting of diabetes transfer.
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Materials and Methods |
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Eight- to 10-wk-old DBA/2 (H-2d) and 4-wk-old NOD (Kd, I-Ag7, Db) mice were purchased from Charles River Breeding Laboratories. Gcn2–/– mice, homozygous for the GCN2 targeted mutation, and wild-type Gcn2+/+ controls were as described (26). NOD-SCID mice were bred in our animal facility under specific pathogen-free conditions to be used at ages 8–10 wk. Transgenic 8.3-NOD mice (27) and BDC2.5-NOD mice (28) were as described. Colorimetric strips were used to monitor glycosuria and hyperglycemia (>300 mg/dl) in NOD-SCID mice (4). Only female mice were used in this study, and all in vivo experiments were done in compliance with National and Perugia University Animal Care and Use Committee guidelines.
Reagents and treatments
The IDO inhibitor 1-methyl-DL-tryptophan (1-MT) was purchased from Sigma-Aldrich to be used in vitro at the concentration of 20 µM. To inhibit IDO activity in vivo, 1-MT or placebo pellets (150 mg/pellet, 7-day release at 0.9 mg/h; Innovative Research of America) were implanted as described (4). NRP-A7 (KYNKANAFL), a synthetic peptide mimotope recognized in the context of the MHC class I H-2Kd molecule by 8.3-CD8+ T cells (29), was as described (13). FITC-conjugated NRP-A7 pentamer and negative control peptide pentamer were prepared by ProImmune and validated by staining 8.3-NOD spleen cells according to manufacturer’s instructions. Pentamer-positive 8.3-CD8+ T cells were recovered by magnetic-activated sorting using anti-FITC MicroBeads (Miltenyi Biotec). Ab reagents for ELISAs of IL-2, IL-4, IL-10, and IFN- have been described (14). A specific TGF- ELISA kit was purchased from Promega. Blockade in vitro of IL-10 and CTLA-4 was achieved by the respective use of SXC-1 (rat IgM) and 4F10 (hamster IgG) monoclonals (13). Purified anti-TGF- 2G7 (mouse IgG2b) was a gift from M. Kopf (Swiss Federal Institute of Technology, Zurich, Switzerland). Neutralizing Abs were added at 10 (anti-IL-10) or 50 µg/ml (anti-CTLA-4, anti-TGF-) and isotype controls were included in all assays. Standard IMDM (Invitrogen Life Technologies), containing 50 µM tryptophan, and low tryptophan (LT, 5 µM) medium (30), both with and without kynurenines (17), were used as culture media. Tryptophan and kynurenine concentrations were measured by HPLC (13).
Cell sorting and suppression assay
CD11c+CD8+ splenic DCs were purified by the sequential use of CD11c and CD8
MicroBeads (Miltenyi Biotec), as described (13), and were referred to as IDO– DCs to distinguish them from IDO+ DCs, i.e., CD11c+CD8+ cells treated overnight with 200 U/ml IFN- so as to induce expression of IDO transcript, protein, and function (4). CD4+CD25+ and CD4+CD25– T cells were isolated from lymph nodes by magnetic-activated cell sorting as described (13, 14). The purity of either T cell fraction was >95%. For assay of regulatory activity, CD4+CD25– cells were cocultured with irradiated T cell-depleted splenocytes and CD4+CD25+ cells for 3 days in the presence of soluble anti-CD3 (1 µg/ml) (14). Proliferation was measured by incorporation of [3H]thymidine according to standard procedures.
Flow cytometry, pentamer staining, and immunoblot analysis
In all FACS analyses, cells were treated with rat anti-CD16/32 (2.4G2) for 30 min at 4°C for blockade of Fc receptors before assaying on an EPICS flow cytometer using EXPO 32 ADC software (Beckman Coulter). The following FITC-labeled Abs were used in cell surface staining (BD Pharmingen): anti-CD4 (GK1.5), anti-CD69 (H1.2F3), and anti-CD45RB (16A). FITC-labeled anti-Foxp3 (FJK-16s) was from eBioscience. PE-labeled anti-CD62L (MEL-14; BD Pharmingen), anti-glucocorticoid-inducible TNFR (GITR, 108619; R&D Systems), and anti-CD4 (GK1.5; BD Pharmingen) were also used. Detection of the surface molecule B and T cell lymphocyte attenuator (BTLA) was accomplished by the use of biotinylated anti-BTLA (a gift from K. Murphy, Washington University, St. Louis, MO) and Streptavidin-FITC. Total CTLA-4 expression was analyzed as described (13). For pentamer staining, spleen and lymph node cells were incubated for 30 min at room temperature with FITC-conjugated H-2Kd pentamers before FACS analysis. For intracellular -chain staining, cells were washed and fixed for 5 min with 2% paraformaldehyde at room temperature, permeabilized and incubated for 30 min with PE-labeled anti-CD3 (6B10.2; Santa Cruz Biotechnology). All FACS analyses included the use of isotype control Abs. For CFSE or CellTrace Far Red DDAO-SE (Molecular Probes) labeling, the fluorescent dyes in the form of 5 mM stock solutions were added to 2 x 107 CD4+ T cells/ml to a final concentration of 2 (CFSE) or 1 µM (DDAO-SE). The cells were incubated at room temperature for 10 min and washed twice with the same volume of FCS and then with culture medium. Immunoblot of TCR -chain and 
-chain involved total T cells or CD4+ and CD8+ fractions purified by magnetic-activated sorting (Miltenyi Biotec), which were analyzed as described (18), using monoclonal anti-CD3 (6B10.2) and polyclonal anti-CD3 (M20; both from Santa Cruz Biotechnology) in combination with the appropriate Abs conjugated to HRP, followed by ECL.
Analysis of lymphocyte function
For mitogen-induced cell proliferation and cytokine induction, CD4+ or CD8+ T cells (1 x 106/ml) were cultured in the presence of 1 µg/ml plate-bound anti-CD3 (145-2C11), 2.5 µg/ml Con A (Sigma-Aldrich), or 2 ng/ml PMA (Sigma-Aldrich) combined with 0.2 µM calcium ionophore (Sigma-Aldrich). In T cell-DC cocultures, T cells (1 x 106) were stimulated with DCs (at a DC to T cell ratio of 1:5 and in the presence of 1 µg/ml soluble anti-CD3), and cultures were assayed for T cell proliferation by incorporation of [3H]thymidine or cell-free supernatants were analyzed for kynurenine or cytokine contents. The lytic activity of CD8+ T cells against 51Cr-labeled, NRP-A7-pulsed RMA-S/Kd target cells was measured as described (29).
Foxp3 expression
Real-time PCR were run on a Chromo4 Four-Color Real-Time Detector (Bio-Rad) using Foxp3-specific primers (5'-CCCAGGAAAGACAGCAACCTT-3'; 5'-TTCTCACAACCAGGCCACTTG-3'). Gapdh (5'-GCCTTCCGTGTTCCTACCC-3'; 5'-CAGTGGGCCCTCAGATGC-3') was the normalizer gene. Conventional RT-PCR was performed using the following primers: Foxp3, 5'-CAGCTGCCTACAGTGCCCCTAG-3' and 5'-CATTTGCCAGCAGTGGGTAG-3'; Gapdh, 5'-AGCACAGTCCATGCCATCAC-3' and 5'-TCCACCACCTGTTGCTGTA-3'. Specific T cell populations were permeabilized and stained with FITC-labeled anti-Foxp3 according to the manufacturer’s instructions.
Statistical analysis
In all assays, the Student’s t test was used for analysis of in vitro and in vivo data.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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-chain in CD8+ T cells
Arginine consumption by macrophages has been reported to modulate the expression of TCR -chain (CD3) in T lymphocytes in the absence of apoptosis (31). We have previously shown that, contrary to Th1 cells, naive T cells do not undergo apoptosis when cultured with IDO+ DCs for 48 h (17). We assessed CD3 expression in CD4+ and CD8+ T lymphocytes from naive mice after culturing these cells with IDO+ DCs in the presence or absence of 1-MT, a specific enzyme inhibitor (Fig. 1A). FACS analysis revealed a noticeable decrease of CD3 in CD8+ T cells cultured with DCs in the absence of 1-MT. Under these conditions, the initial 50 µM tryptophan concentration dropped to 
5 µM by 24 h, a time when the concentrations of the major tryptophan catabolites L-kynurenine, 3-hydroxykynurenine, 3-hydroxyanthranilic acid (3-HAA), and quinolinic acid were in the low micromolar range (Fig. 1B).
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-chain down-regulation occurred by the direct action of tryptophan catabolism and to identify the possible milieu responsible for this effect, we cultured CD4+ and CD8+ T cells in medium containing a LT (5 µM) concentration, a mixture of 3-hydroxykynurenine, 3-HAA, and quinolinic acid, each at 10 µM, or a combination of LT with kynurenines (LT-K). Plate-bound anti-CD3 and anti-CD28 were present in the cultures. FACS analysis revealed that the LT-K culture conditions, but not LT or kynurenines alone, decreased CD3, and did so mostly in CD8+ T cells (Fig. 1C). The LT-K conditions had no effect on the expression of other TCR components, including TCR and TCR, and did not affect costimulatory molecules, including CD28 (data not shown). The effect of LT-K was time-dependent (Fig. 1D) and slowly reversible, as it required reculturing the cells for at least 72 h in control medium for 90% recovery of the original expression (data not shown). The effect was also demonstrable by immunoblot analysis with anti- but not anti- Abs, and was likewise observable using IDO+ DCs in place of LT-K (Fig. 1E).
The GCN2 stress-response kinase is activated by starvation for amino acids and functions to control transcriptional and translational programs that couple cell growth to amino acid availability (26). Recent evidence indicates that GCN2 activation could be an upstream mechanism in the immunoregulatory effects of IDO (32). To ascertain whether down-regulation of CD3 by tryptophan catabolism requires GCN2, we used Gcn2–/– mice as a source of CD8+ T cells that were exposed to LT-K medium (Fig. 1F). In contrast to the Gcn2+/+ mice, we observed no changes in CD3 expression in the T cells lacking GCN2.
TCR -chain down-regulation is associated with impaired function
We investigated whether the diminished expression of CD3 induced by LT and kynurenines in CD8+ T cells correlates with an impaired immune function in vitro. Purified CD8+ and CD4+ T cells were exposed to LT-K for 72 h, washed and recultured for 48 h in the presence of anti-CD3, Con A, or a combination of PMA and calcium ionophore (Fig. 2A). Proliferation was significantly reduced by LT-K conditioning upon activation of CD8+ cells, but not CD4+ cells, with anti-CD3 or Con A. No effect was found on activation with PMA and calcium ionophore in either cell type, demonstrating a selective impairment of TCR-mediated signaling events in the CD8+ cells conditioned by LT-K. Under the same conditions, the production of IFN- and IL-2 by CD8+ T cells in response to anti-CD3 was severely impaired (Fig. 2B). The cytokine response of CD4+ T cells was apparently unaffected (data not shown).
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transgene (8.3-NOD) (27) possess a prevalent population of autoreactive CD8+ T cells that recognize a cell autoantigen and are reactive to the islet-related NRP-A7 peptide (29, 33, 34). To ascertain whether -chain down-regulation occurs in vivo, we used pentamer technology to transfer NOD-SCID mice with a combination of NRP-A7-pulsed, IDO+ DCs and NRP-A7 pentamer-positive 8.3-CD8+ T cells. The CD8+ cells were harvested from the spleen and lymph nodes, and were analyzed for CD3 expression (Fig. 2C) and for ability to lyse peptide-pulsed target cells (Fig. 2D). Noticeable down-regulation of the -chain and impaired lytic activity were observed in the CD8+ T cells recovered from NOD-SCID mice immunized with IDO+ DCs at the time of T cell transfer. Tryptophan catabolism converts CD25–Foxp3– into CD25+Foxp3+ cells
Despite the lack of LT-K effects on proliferation and cytokine production by CD4+ T cells following a 48-h exposure, we asked whether a prolonged exposure to LT and kynurenines might act on naive CD4+ T cells to change their functional properties. When CD4+ T cells exposed to LT-K for 7 d were recultured for 24 h with anti-CD3 in standard medium, they produced little or no IFN- and IL-4; yet, they produced noticeable amounts of IL-10 and TGF- (Fig. 3). A similar pattern of cytokine production was observed on assaying CD4+ T cells exposed to IDO+ DCs in place of LT-K (data not shown).
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secretion, represent a dedicated T cell lineage, and that the forkhead family transcription factor Foxp3 functions as the Treg cell lineage specification factor (35). We used real-time PCR analysis for measuring Foxp3 transcript expression in unfractionated CD4+ T cells exposed to IDO– or IDO+ DCs or to LT or LT-K for 1, 3, or 6 days (Fig. 4A). The results demonstrated an 8- to 40-fold increase in Foxp3 transcription at 3–6 days of cell culture with IDO+ DCs or in the LT-K medium. The effect of culturing CD4+ T cells with IDO+ DCs on Foxp3 transcript expression was negated by the presence of 1-MT.
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We used FACS analysis to investigate the possible conversion of CD25– cells into CD25+ cells in concomitance with Foxp3 appearance, and to evaluate the expression of a series of phenotypic and functional markers including CD69, CD45RB, CD62L, CTLA-4, BTLA, and GITR. At 1 wk of LT-K exposure, >90% of the original CD25– cells appeared to express the CD25 molecule. The converted cells were CD69–, CD45RBlow, CD62L+, CTLA-4+, BTLAlow, and GITR+, thus expressing markers associated with the function of CD4+CD25+ Tregs and subsets thereof in different experimental settings (28, 36, 37, 38, 39) (Fig. 4D).
Next we investigated whether the conversion of CD4+CD25– T cells into CD4+CD25+ Tregs by LT-K could be the result of expansion of residual CD4+CD25+ cells or selective CD4+CD25+ cell survival. Using individual populations or combinations of CD4+CD25– and CD4+CD25+ T cells we examined proliferation, cell division, and viable cell recovery data at different times of LT-K exposure. Purified CD4+CD25– T cells but not CD4+CD25+ proliferated in LT-K medium, by both [3H]thymidine uptake and trypan blue dye exclusion analysis (Fig. 5A). Coculturing CFSE-labeled CD4+CD25– cells with 10% DDAO-SE-labeled CD4+CD25+ cells in LT-K revealed selective proliferation of CD4+CD25– cells (Fig. 5B). Thus, neither expansion of residual CD4+CD25+ cells nor selective CD4+CD25+ cell survival contributed significantly to the conversion of CD4+CD25– T cells into CD4+CD25+ Tregs by LT-K.
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To investigate whether the CD4+CD25+ Foxp3-expressing cells induced by LT-K display regulatory function in vitro, we cocultured various numbers of those cells with responder CD4+CD25– T lymphocytes from naive mice in the presence of anti-CD3 and T cell-depleted splenocyte samples as APCs (Fig. 6A). CD4+CD25– cells were exposed to LT-K or cultured with IDO+ DCs for 7 days (a time when no viable CD8+ T cells would be recoverable from parallel cultures; Fig. 5A), and cells were then assayed for suppressive activity after separation into CD25+ and CD25– fractions. The CD4+CD25+ T cells cultured in LT-K or with IDO+ DCs for 7 days greatly inhibited the proliferation of responder T cells in contrast to the minority fraction that had remained CD25– after LT-K or IDO+ DC treatment. (These cells did not express Foxp3 transcripts, data not shown.) Suppression was comparable to that of natural Tregs, i.e., CD4+CD25+ Foxp3-expressing T cells isolated from lymph nodes of naive mice.
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, or IL-10 has any role in mediating the regulatory function of the CD25+ cells induced by LT-K. Abs to CTLA-4, TGF-, or IL-10 were added, either singly or in combination, to the cocultures of Treg and responder CD25– T cells in the proliferation assay (Fig. 6B). The Abs had limited (anti-IL-10, anti-CTLA-4) or no (anti-TGF-) effect when used singly, but the combination of anti-IL-10 and anti-CTLA-4 resulted in ablation of the regulatory properties of the Tregs generated by LT-K exposure.
GCN2 and TGF- are required for generation of Foxp3+ Tregs by LT-K
Naive CD4+CD25– T cells can be converted to a CD25+ Treg phenotype by TGF-, which is thought to act through the induction of Foxp3 (37). Although the production of this cytokine seemed to have little role in the in vitro downstream effects of the T cells converted by LT-K, we asked whether autocrine TGF- is involved in Foxp3 induction. We also examined whether activation of the GCN2 kinase is a prerequisite for the induction of Foxp3 and Treg function by LT-K. Gcn2–/– and wild-type mice served as a source of CD4+CD25– cells that were used for generating Tregs by 7-day exposure to LT-K in the presence of anti-CD3 and anti-CD28, with or without anti-TGF-. Foxp3 transcript expression (Fig. 6C) and ability to inhibit the proliferation of responder T cells (Fig. 6D) were examined in the recovered cells according to the conditions described above. The GCN2 kinase and TGF- appeared to be required for the expression of Foxp3 and regulatory properties in CD4+ T cells exposed to LT-K. Although it is possible that the anti-TGF- would also neutralize any TGF- present in the bovine sera added to the media, it should be noticed that the DCs did not produce significant amounts of TGF- in vitro in response to LT-K (data not shown).
Tregs induced by LT-K protect hosts from diabetes transfer
Clonotypic T cell NOD mice expressing the BDC2.5-TCR transgene (BDC2.5/NOD) are characterized by a prevalent population of CD4+ T cells that recognize a mimotope homologous to a peptide of glutamic acid decarboxylase 65 (40, 41, 42). Most BDC2.5/NOD mice do not develop spontaneous diabetes, but do develop insulitis accompanied by the appearance of activated islet-specific T cells. Evidence suggests that these cells are actively prevented from causing disease by a CTLA-4-dependent immunoregulatory mechanism and by the action of CD4+CD25+CD69– Tregs (43). At variance with BDC2.5/NOD mice, BDC2.5 TCR transgenic mice backcrossed into the NOD-SCID background show fulminant diabetes, strongly arguing for the regulatory role of endogenously rearranged T cells lacking the transgenic TCR (44). Rapid diabetes also develops in NOD-SCID mice adoptively transferred with spleen cells or purified CD4+ T cells from BDC2.5/NOD donors (45). In this setting, however, onset of disease is prevented by the cotransfer of CD4+CD25+CD62L+ cells from prediabetic NOD mice (45). Using the cotransfer model, we investigated whether the Tregs generated by LT-K in vitro are capable of protecting NOD-SCID mice from the induction of diabetes by BDC2.5 transgenic T cells. We tested CD4+CD25– cells cultured for 7 d in LT-K medium in comparison with purified CD4+CD25+CD62L+ cells from NOD mice (Fig. 7A). The results showed that the Tregs generated by LT-K prevented transfer of diabetes in a durable and dose-dependent fashion and to an extent comparable to the control Treg population. However, when the recipients of the cotransfer received 1-MT pellets at the time of cell transfer, neither the CD4+CD25+ Treg population generated by LT-K nor the control Treg population was capable of protecting the hosts from diabetes transfer, and the proportions of disease-free animals at 30 days were 0% and 25% for the two treatments, respectively (Fig. 7B). This result demonstrates that the Tregs induced by LT-K protect NOD-SCID mice from diabetes transfer via IDO-dependent mechanisms, and so does the CD25+CD62L+ subset of Tregs from early prediabetic NOD mice.
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Presumably due to their expression of tryptophanyl-tRNA-synthetase, which counteracts IDO-induced tryptophan depletion, naive CD8+ T cells are resistant to cell cycle arrest by tryptophan catabolism when exposed to strong antigenic stimuli (15), and are likewise resistant to kynurenine-induced apoptosis (17). Yet, Ag-specific class I-restricted responses are severely and reversibly impaired under conditions of IDO activation in vivo (10, 11). TCR -chain down-regulation and impaired in vitro T cell function have been described in cancer and autoimmune and infectious diseases (19). Although down-regulation of -chain could be a normal mechanism for controlling an excessive and potentially hazardous immune response, the continuous presence of pathogen or tumor Ags and chronic inflammation most likely exploits this mechanism to impair immune responses to chronic diseases (18), many of which are characterized by sustained IDO expression (1, 2). We found that a LT concentration, incapable of impairing T cell reactivity per se (30), synergized with physiologic concentrations of kynurenines (17) in down-regulating T cell CD3 expression in a specific and reversible fashion. The effect was demonstrable in vivo and required the activity of the GCN2 kinase. Although a causal link was not established between down-regulation of CD3 expression and impaired function of CD8+ T cells, the data suggested an association of the former effect with a reduced cytotoxic activity in vitro.
In eukaryotes, the mechanism for recognizing indispensable amino acid deficiency follows the conserved general control system, wherein uncharged transfer RNA induces phosphorylation of eukaryotic initiation factor 2 via the GCN2 kinase. This basic mechanism of nutritional stress management functions primarily to couple cell growth to amino acid availability (26). It has been proposed that GCN2 acts as a molecular sensor in T cells, allowing them to detect and respond to conditions created by IDO (32). Although amino acid deficiency alone is necessary and sufficient to activate a GCN2 response, our current data demonstrate that CD3 down-regulation requires the combined effects of tryptophan depletion and tryptophan catabolites. This clarifies the qualitative nature of the stress conditions created by IDO in vivo and, at the same time, explains the specificity of the T cell response to this particular form of ‘nutritional’ stress. In the case of conditions generated by tumoral IDO (11), the exquisite sensitivity of TCR-expressing T cells to LT-K effects could explain why tryptophan catabolism likely represents an efficient mechanism used by tumors for immune evasion in the absence of any gross effect of nutrient depletion, which would be detrimental to the tumor itself.
CD4+CD25+ Tregs are crucial in controlling autoimmune and inflammatory pathology, but little is known about their development and maintenance (20, 21, 22, 23). Although it appears very likely that most CD4+CD25+ Treg cell development occurs in the thymus, accumulating evidence suggests that these cells can also develop in the periphery (50, 51), and that they do so under natural conditions, i.e., with the expression of a natural TCR repertoire and exposure to the natural endogenous Ag load (38). We found that mature peripheral CD4+CD25– T cells, that are relatively resistant to CD3 down-regulation by LT-K, can indeed convert to a CD4+CD25+ Treg phenotype through a process requiring GCN2 and leading to a gradual decrease in IL-2 production and up-regulation of IL-10 and TGF-. Although the converted CD4+CD25+ cells themselves fail to proliferate after stimulation, they can suppress proliferation of responder cells in vitro. The converted cells express high amounts of Foxp3 transcript and Foxp3 protein. Autocrine TGF- appears to be a prerequisite for the induction of Foxp3 and Treg function.
Much evidence is now available for the diversity of CD4+ Tregs that control distinct pathologies (52). Treg subsets defined by the CD25, CD62L, and CD45RB markers overlap only partially, suggesting that these subsets are functionally distinct or that a relevant Treg subset represents only a minority of the cells composing the three populations defined by these markers. As a result, the Treg population that protects NOD mice from fulminant diabetes appears to be CD25+CD62L+, shows a variable expression of CD45RB, and is distinct from the Treg subset protecting from colitis or gastritis (28). Conversion of peripheral CD4+CD25– naive T cells to CD4+CD25+ Tregs by TGF- induction of Foxp3 results in the appearance of a CD25+CD45RBlowCTLA-4+ subset that is unresponsive to TCR stimulation, produces neither Th1 nor Th2 cytokines, expresses TGF-, and inhibits normal T cell proliferation in vitro (37). Similar to this Treg population, we found that cell conversion in our model system with LT-K was further characterized by the presence of CD62L, GITR and secreted IL-10, with little or no expression of CD69 or BTLA.
Among Foxp3-expressing Treg populations, CD4+CD25+ T cells are known to function through different mechanisms, including cell-cell contact, membrane or soluble TGF-, and secreted IL-10 (21, 23, 52). We found that the Tregs generated by LT and kynurenines would suppress naive T cell proliferation in vitro by a combination of CTLA-4-dependent and IL-10-dependent effects, as demonstrated by the blocking effect of neutralizing Abs. In vivo, we found that the CD4+CD25+ Tregs induced by LT-K prevented transfer of diabetes to an extent comparable to the control Treg population from prediabetic NOD mice. The protection transferred by both populations required tryptophan catabolism in the host. This indicates that modulation of tryptophan catabolism could represent an important mechanism of action not only for developmentally controlled ‘professional’ Tregs (52), as suggested by previous data (13), but also for T cells that are induced in the periphery to express a regulatory phenotype.
In conclusion, our data demonstrate that, before T cell cycle arrest due to a critical shortage in tryptophan supply, the early control of an immune response by IDO involves adjustable and versatile effects, including CD3 down-regulation and Treg generation, operated by two distinct signals, i.e., tryptophan deficiency and kynurenine production. Although the mechanisms of kynurenine action are largely unknown (17, 25, 53, 54), this observation clearly distinguishes the activity of IDO as a truly immunoregulatory enzyme from the effects on T cells of a mere starvation for amino acids, and paves the way to the possible use of synthetic kynurenines as therapeutic agents. Defining one possible means by which Tregs are physiologically generated and maintained in the periphery could provide new opportunities for designing ways to induce or abrogate tolerance in physiopathologic conditions.
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Acknowledgments |
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Disclosures |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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Footnotes |
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1 This work was supported by the Italian Association for Cancer Research. P.S. is supported by grants from the Canadian Institutes of Health Research and is a Scientist of the Alberta Heritage Foundation for Medical Research. 
2 Address correspondence and reprint requests to Dr. Paolo Puccetti, Department of Experimental Medicine, Section of Pharmacology, University of Perugia, Via del Giochetto, Perugia 06126, Italy. E-mail address: plopcc{at}tin.it
3 Abbreviations used in this paper: DC, dendritic cell; BTLA, B and T cell lymphocyte attenuator; GITR, glucocorticoid-inducible TNFR; 3-HAA, 3-hydroxyanthranilic acid; LT, low tryptophan; LT-K, LT with kynurenine; 1-MT, 1-methyl-DL-tryptophan; Treg, regulatory T cell.
Received for publication January 26, 2006. Accepted for publication March 16, 2006.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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-dependent T cell receptor down-regulation and impaired T cell function. Nat. Immunol. 4: 957-964. [Medline]-chain downregulation: curtailing an excessive inflammatory immune response. Nat. Rev. Immunol. 4: 675-687. [Medline] kinase is required for adaptation to amino acid deprivation in mice. Mol. Cell. Biol. 22: 6681-6688. cell antigen targeted by a prevalent population of pathogenic CD8+ T cells in autoimmune diabetes. Proc. Natl. Acad. Sci. USA 100: 8384-8388. chain expression by L-arginine. J. Biol. Chem. 277: 21123-21129. induction of transcription factor Foxp3. J. Exp. Med. 198: 1875-1886. genes. J. Immunol. 166: 2982-2991. cell apoptosis in T cell-mediated autoimmune diabetes. Proc. Natl. Acad. Sci. USA 94: 213-218. Related articles in The JI:
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N. A. Barnes, S. J. Stephenson, R. M. Tooze, and G. M. Doody Amino Acid Deprivation Links BLIMP-1 to the Immunomodulatory Enzyme Indoleamine 2,3-Dioxygenase J. Immunol., November 1, 2009; 183(9): 5768 - 5777. [Abstract] [Full Text] [PDF]
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 F. Fallarino, G. Luca, M. Calvitti, F. Mancuso, C. Nastruzzi, M. C. Fioretti, U. Grohmann, E. Becchetti, A. Burgevin, R. Kratzer, et al. Therapy of experimental type 1 diabetes by isolated Sertoli cell xenografts alone J. Exp. Med., October 26, 2009; 206(11): 2511 - 2526. [Abstract] [Full Text] [PDF]
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 B. Jurgens, U. Hainz, D. Fuchs, T. Felzmann, and A. Heitger Interferon-{gamma}-triggered indoleamine 2,3-dioxygenase competence in human monocyte-derived dendritic cells induces regulatory activity in allogeneic T cells Blood, October 8, 2009; 114(15): 3235 - 3243. [Abstract] [Full Text] [PDF]
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 C Coquerelle, G Oldenhove, V Acolty, J Denoeud, G Vansanten, J-M Verdebout, A Mellor, J A Bluestone, and M Moser Anti-CTLA-4 treatment induces IL-10-producing ICOS+ regulatory T cells displaying IDO-dependent anti-inflammatory properties in a mouse model of colitis Gut, October 1, 2009; 58(10): 1363 - 1373. [Abstract] [Full Text] [PDF]
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 K. C. McKenna, K. M. Beatty, R. A. Bilonick, L. Schoenfield, K. L. Lathrop, and A. D. Singh Activated CD11b+ CD15+ Granulocytes Increase in the Blood of Patients with Uveal Melanoma Invest. Ophthalmol. Vis. Sci., September 1, 2009; 50(9): 4295 - 4303. [Abstract] [Full Text] [PDF]
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 S. P. Cobbold, E. Adams, C. A. Farquhar, K. F. Nolan, D. Howie, K. O. Lui, P. J. Fairchild, A. L. Mellor, D. Ron, and H. Waldmann Infectious tolerance via the consumption of essential amino acids and mTOR signaling PNAS, July 21, 2009; 106(29): 12055 - 12060. [Abstract] [Full Text] [PDF]
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D. J. Chung, M. Rossi, E. Romano, J. Ghith, J. Yuan, D. H. Munn, and J. W. Young Indoleamine 2,3-dioxygenase-expressing mature human monocyte-derived dendritic cells expand potent autologous regulatory T cells Blood, July 16, 2009; 114(3): 555 - 563. [Abstract] [Full Text] [PDF]
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H. Liu, L. Liu, K. Liu, P. Bizargity, W. W. Hancock, and G. A. Visner Reduced Cytotoxic Function of Effector CD8+ T Cells Is Responsible for Indoleamine 2,3-Dioxygenase-Dependent Immune Suppression J. Immunol., July 15, 2009; 183(2): 1022 - 1031. [Abstract] [Full Text] [PDF]
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M. Brenk, M. Scheler, S. Koch, J. Neumann, O. Takikawa, G. Hacker, T. Bieber, and D. von Bubnoff Tryptophan Deprivation Induces Inhibitory Receptors ILT3 and ILT4 on Dendritic Cells Favoring the Induction of Human CD4+CD25+ Foxp3+ T Regulatory Cells J. Immunol., July 1, 2009; 183(1): 145 - 154. [Abstract] [Full Text] [PDF]
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G. N. Scott, J. DuHadaway, E. Pigott, N. Ridge, G. C. Prendergast, A. J. Muller, and L. Mandik-Nayak The Immunoregulatory Enzyme IDO Paradoxically Drives B Cell-Mediated Autoimmunity J. Immunol., June 15, 2009; 182(12): 7509 - 7517. [Abstract] [Full Text] [PDF]
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 Y. H. Kim, B. K. Choi, W. J. Kang, K. H. Kim, S. W. Kang, A. L. Mellor, D. H. Munn, and B. S. Kwon IFN-{gamma}-indoleamine-2,3 dioxygenase acts as a major suppressive factor in 4-1BB-mediated immune suppression in vivo J. Leukoc. Biol., May 1, 2009; 85(5): 817 - 825. [Abstract] [Full Text] [PDF]
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 J. Correale, M. C. Ysrraelit, and M. I. Gaitan Immunomodulatory effects of Vitamin D in multiple sclerosis Brain, May 1, 2009; 132(5): 1146 - 1160. [Abstract] [Full Text] [PDF]
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 A. Becerra, R. V. Warke, K. Xhaja, B. Evans, J. Evans, K. Martin, N. de Bosch, A. L. Rothman, and I. Bosch Increased activity of indoleamine 2,3-dioxygenase in serum from acutely infected dengue patients linked to gamma interferon antiviral function J. Gen. Virol., April 1, 2009; 90(4): 810 - 817. [Abstract] [Full Text] [PDF]
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A. Boasso, M. Vaccari, D. Fuchs, A. W. Hardy, W.-P. Tsai, E. Tryniszewska, G. M. Shearer, and G. Franchini Combined Effect of Antiretroviral Therapy and Blockade of IDO in SIV-Infected Rhesus Macaques J. Immunol., April 1, 2009; 182(7): 4313 - 4320. [Abstract] [Full Text] [PDF]
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A. Curti, S. Trabanelli, V. Salvestrini, M. Baccarani, and R. M. Lemoli The role of indoleamine 2,3-dioxygenase in the induction of immune tolerance: focus on hematology Blood, March 12, 2009; 113(11): 2394 - 2401. [Abstract] [Full Text] [PDF]
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 A. L. Putnam, T. M. Brusko, M. R. Lee, W. Liu, G. L. Szot, T. Ghosh, M. A. Atkinson, and J. A. Bluestone Expansion of Human Regulatory T-Cells From Patients With Type 1 Diabetes Diabetes, March 1, 2009; 58(3): 652 - 662. [Abstract] [Full Text] [PDF]
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 R. B. Jalili, F. Forouzandeh, A. Moeenrezakhanlou, G. R. Rayat, R. V. Rajotte, H. Uludag, and A. Ghahary Mouse Pancreatic Islets Are Resistant to Indoleamine 2,3 Dioxygenase-Induced General Control Nonderepressible-2 Kinase Stress Pathway and Maintain Normal Viability and Function Am. J. Pathol., January 1, 2009; 174(1): 196 - 205. [Abstract] [Full Text] [PDF]
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C. Orabona, M. T. Pallotta, C. Volpi, F. Fallarino, C. Vacca, R. Bianchi, M. L. Belladonna, M. C. Fioretti, U. Grohmann, and P. Puccetti SOCS3 drives proteasomal degradation of indoleamine 2,3-dioxygenase (IDO) and antagonizes IDO-dependent tolerogenesis PNAS, December 30, 2008; 105(52): 20828 - 20833. [Abstract] [Full Text] [PDF]
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W. Chen, X. Liang, A. J. Peterson, D. H. Munn, and B. R. Blazar The Indoleamine 2,3-Dioxygenase Pathway Is Essential for Human Plasmacytoid Dendritic Cell-Induced Adaptive T Regulatory Cell Generation J. Immunol., October 15, 2008; 181(8): 5396 - 5404. [Abstract] [Full Text] [PDF]
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A. Das, M. Hoare, N. Davies, A. R. Lopes, C. Dunn, P. T.F. Kennedy, G. Alexander, H. Finney, A. Lawson, F. J. Plunkett, et al. Functional skewing of the global CD8 T cell population in chronic hepatitis B virus infection J. Exp. Med., September 1, 2008; 205(9): 2111 - 2124. [Abstract] [Full Text] [PDF]
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 L. Harrington, C. V. Srikanth, R. Antony, S. J. Rhee, A. L. Mellor, H. N. Shi, and B. J. Cherayil Deficiency of Indoleamine 2,3-Dioxygenase Enhances Commensal-Induced Antibody Responses and Protects against Citrobacter rodentium-Induced Colitis Infect. Immun., July 1, 2008; 76(7): 3045 - 3053. [Abstract] [Full Text] [PDF]
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L. Romani, T. Zelante, A. De Luca, F. Fallarino, and P. Puccetti IL-17 and Therapeutic Kynurenines in Pathogenic Inflammation to Fungi J. Immunol., April 15, 2008; 180(8): 5157 - 5162. [Abstract] [Full Text] [PDF]
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 M. Razmara, B. Hilliard, A. K. Ziarani, Y. H. Chen, and M. L. Tykocinski CTLA-4{middle dot}Ig converts naive CD4+CD25- T cells into CD4+CD25+ regulatory T cells Int. Immunol., April 1, 2008; 20(4): 471 - 483. [Abstract] [Full Text] [PDF]
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L. K. Jasperson, C. Bucher, A. Panoskaltsis-Mortari, P. A. Taylor, A. L. Mellor, D. H. Munn, and B. R. Blazar Indoleamine 2,3-dioxygenase is a critical regulator of acute graft-versus-host disease lethality Blood, March 15, 2008; 111(6): 3257 - 3265. [Abstract] [Full Text] [PDF]
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C. H. Cook, A. A. Bickerstaff, J.-J. Wang, T. Nadasdy, P. Della Pelle, R. B. Colvin, and C. G. Orosz Spontaneous Renal Allograft Acceptance Associated with "Regulatory" Dendritic Cells and IDO J. Immunol., March 1, 2008; 180(5): 3103 - 3112. [Abstract] [Full Text] [PDF]
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 S. Li, E. J. Gowans, C. Chougnet, M. Plebanski, and U. Dittmer Natural Regulatory T Cells and Persistent Viral Infection J. Virol., January 1, 2008; 82(1): 21 - 30. [Full Text] [PDF]
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 J. Li, A. Meinhardt, M.-E. Roehrich, D. Golshayan, J. Dudler, M. Pagnotta, M. Trucco, and G. Vassalli Indoleamine 2,3-dioxygenase gene transfer prolongs cardiac allograft survival Am J Physiol Heart Circ Physiol, December 1, 2007; 293(6): H3415 - H3423. [Abstract] [Full Text] [PDF]
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A. De Luca, C. Montagnoli, T. Zelante, P. Bonifazi, S. Bozza, S. Moretti, C. D'Angelo, C. Vacca, L. Boon, F. Bistoni, et al. Functional yet Balanced Reactivity to Candida albicans Requires TRIF, MyD88, and IDO-Dependent Inhibition of Rorc J. Immunol., November 1, 2007; 179(9): 5999 - 6008. [Abstract] [Full Text] [PDF]
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S. W. Tas, M. J. Vervoordeldonk, N. Hajji, J. H. N. Schuitemaker, K. F. van der Sluijs, M. J. May, S. Ghosh, M. L. Kapsenberg, P. P. Tak, and E. C. de Jong Noncanonical NF-{kappa}B signaling in dendritic cells is required for indoleamine 2,3-dioxygenase (IDO) induction and immune regulation Blood, September 1, 2007; 110(5): 1540 - 1549. [Abstract] [Full Text] [PDF]
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 R. Metz, J. B. DuHadaway, U. Kamasani, L. Laury-Kleintop, A. J. Muller, and G. C. Prendergast Novel Tryptophan Catabolic Enzyme IDO2 Is the Preferred Biochemical Target of the Antitumor Indoleamine 2,3-Dioxygenase Inhibitory Compound D-1-Methyl-Tryptophan Cancer Res., August 1, 2007; 67(15): 7082 - 7087. [Abstract] [Full Text] [PDF]
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A. P. J. van der Marel, J. N. Samsom, M. Greuter, L. A. van Berkel, T. O'Toole, G. Kraal, and R. E. Mebius Blockade of IDO Inhibits Nasal Tolerance Induction J. Immunol., July 15, 2007; 179(2): 894 - 900. [Abstract] [Full Text] [PDF]
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H. Hamdi, V. Godot, M.-C. Maillot, M. V. Prejean, N. Cohen, R. Krzysiek, F. M. Lemoine, W. Zou, and D. Emilie Induction of antigen-specific regulatory T lymphocytes by human dendritic cells expressing the glucocorticoid-induced leucine zipper Blood, July 1, 2007; 110(1): 211 - 219. [Abstract] [Full Text] [PDF]
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Z. Zhang, C. L. Gorman, A.-C. Vermi, C. Monaco, A. Foey, S. Owen, P. Amjadi, A. Vallance, C. McClinton, F. Marelli-Berg, et al. TCR{zeta}dimlymphocytes define populations of circulating effector cells that migrate to inflamed tissues Blood, May 15, 2007; 109(10): 4328 - 4335. [Abstract] [Full Text] [PDF]
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A. Boasso, J.-P. Herbeuval, A. W. Hardy, S. A. Anderson, M. J. Dolan, D. Fuchs, and G. M. Shearer HIV inhibits CD4+ T-cell proliferation by inducing indoleamine 2,3-dioxygenase in plasmacytoid dendritic cells Blood, April 15, 2007; 109(8): 3351 - 3359. [Abstract] [Full Text] [PDF]
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E. Larrea, J. I. Riezu-Boj, L. Gil-Guerrero, N. Casares, R. Aldabe, P. Sarobe, M. P. Civeira, J. L. Heeney, C. Rollier, B. Verstrepen, et al. Upregulation of Indoleamine 2,3-Dioxygenase in Hepatitis C Virus Infection J. Virol., April 1, 2007; 81(7): 3662 - 3666. [Abstract] [Full Text] [PDF]
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Z. Liu, H. Dai, N. Wan, T. Wang, S. Bertera, M. Trucco, and Z. Dai Suppression of Memory CD8 T Cell Generation and Function by Tryptophan Catabolism J. Immunol., April 1, 2007; 178(7): 4260 - 4266. [Abstract] [Full Text] [PDF]
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A. Curti, S. Pandolfi, B. Valzasina, M. Aluigi, A. Isidori, E. Ferri, V. Salvestrini, G. Bonanno, S. Rutella, I. Durelli, et al. Modulation of tryptophan catabolism by human leukemic cells results in the conversion of CD25- into CD25+ T regulatory cells Blood, April 1, 2007; 109(7): 2871 - 2877. [Abstract] [Full Text] [PDF]
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6), as revealed by glycosuria and hyperglycemia. One representative experiment of three conducted is shown. Breaks in lines and axis indicate scale interruption. B, Naive CD4+CD25– cells were cultured with LT-K for 7 days as indicated in A. Sorted CD25+ cells or CD62L+ T cells from prediabetic NOD mice (2 x 106) were cotransferred with diabetogenic NOD/BDC2.5 splenocytes (1 x 106) into NOD-SCID mice that had been implanted with placebo or 1-MT pellets. The animals were then monitored for diabetes incidence. One of three experiments with similar results.