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Keywords:

  • Dendritic cells;
  • Immunoregulation;
  • Indoleamine 2,3-dioxygenase;
  • Interferon

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements

Following CD80/86 (B7) and TLR9 ligation, small subsets of splenic dendritic cells expressing CD19 (CD19+ DC) acquire potent T cell regulatory functions due to induced expression of the intracellular enzyme indoleamine 2,3-dioxygenase (IDO), which catabolizes tryptophan. In CD19+ DC, IFN type I (IFN-α) is the obligate inducer of IDO. We now report that IFN-α production needed to stimulate high-level expression of IDO following B7 ligation is itself dependent on basal levels of IDO activity. Genetic and pharmacologic ablation of IDO completely abrogated IFN-α production by CD19+ DC after B7 ligation. In contrast, IDO ablation did not block IFN-α production by CD19+ DC after TLR9 ligation. IDO-mediated control of IFN-α production depended on tryptophan depletion as adding excess tryptophan also blocked IFN-α expression after B7 ligation. Consistent with this, DC from mice deficient in general control of non-derepressible-2 (GCN2)-kinase, a component of the cellular stress response to amino acid withdrawal, did not produce IFN-α following B7 ligation, but produced IFN-α after TLR9 ligation. Thus, B7 and TLR9 ligands stimulate IFN-α expression in CD19+ DC via distinct signaling pathways. In the case of B7 ligation, IDO activates cell-autonomous signals essential for IFN-α production, most likely by activating the GCN2-kinase-dependent stress response.

See accompanying commentary: http://dx.doi.org/10.1002/eji.20073737184

Abbreviations:
1mT:

1-methyl-D-tryptophan

B7:

CD80/CD86

CpG-ODN:

synthetic oligonucleotide containing unmethylated CpG motifs

GCN2:

general control of non-derepressible-2

IDO:

indoleamine 2,3-dioxygenase

IFNAR:

IFN-α receptor

ISR:

integrated stress response

pDC:

plasmacytoid DC

Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements

Indoleamine 2,3-dioxygenase (IDO) enzyme activity protects tissues from T cell-mediated immunity during pregnancy and in several autoimmune and allergic disease models 13. IDO activity also inhibits T cell responses to tumor cells 48 and to cells infected with the human immunodeficiency virus (HIV) 9. Thus, IDO is a key component of inflammatory responses that can be either detrimental or beneficial to disease outcomes. Furthermore, manipulating the IDO mechanism using IDO inhibitors or IDO inducers is likely to yield therapeutic effects in a range of human clinical disorders.

IDO expression is controlled by transcriptional and post-translational factors that regulate IDO activity in specific cell types. Interferon type I (IFN-α/β) and type II (IFN-γ) can both induce IDO transcription by activating specific signaling pathways in responsive cell types 1012. As expected, ablating the signal transducer and activator of transcription-1 (STAT-1), which is required for IFN signaling, completely blocked IDO up-regulation in response to CD80/86 (B7) ligation 13. The timing of IFN signaling relative to other DC maturation and activation signals is critical for induction of functional IDO activity in DC 14. We and others have described specific subsets of human and murine DC that up-regulate IDO and acquire potent IDO-dependent T cell regulatory functions when exposed to soluble CTLA4-Ig fusion protein or anti-B7 antibodies that ligate surface B7 molecules 1319. Recently, we reported that high systemic doses of synthetic oligonucleotides containing unmethylated CpG motifs (CpG-ODN) (TLR9 ligands) also induced splenic CD19+ DC to express IDO and acquire T cell suppressive functions 20, and similar findings were reported by Wingender et al.21. In mice, B7 ligands induced IDO exclusively in a small subset of splenic DC located in the splenic red pulp that co-expressed CD8α, the B cell/plasmacytoid DC (pDC) marker B220, and the B cell marker CD19 1820. CD19+ DC resemble conventional pDC (CD11clow B220+ CD19neg) morphologically, as well as functionally since sorted CD19+ DC produced IFN-α following B7 ligation 19. However, CD19+ DC were the only DC subset that responded to B7 or TLR9 ligation by activating STAT-1 and subsequently expressing IDO via IFN-α signaling 19, 20. In the current study, we show that IDO activity is itself essential for IFN-α expression by CD19+ DC following B7 ligation, while IDO activity is not essential for IFN-α expression by CD19+ DC following TLR9 ligation.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements

An intact IDO gene is essential to induce IFN-α after B7 ligation

In previous studies, we reported that upstream IFN-α signaling via IFN-α/β receptors was indispensable for IDO up-regulation following B7 or CpG ligation in CD19+ DC 19, 20. In each case, IDO induction did not occur when IFN-α receptor (IFNAR) signaling was blocked by gene inactivation, or by sequestering IFN-α using anti-IFN-α mAb. In contrast, blocking IFN-γ receptor signaling by genetic ablation, or by sequestering IFN-γ, had no effect of IDO up-regulation in CD19+ DC.

Because IFN-α is the obligate upstream inducer of IDO in CD19+ DC, we were surprised to discover that CTLA4-Ig treatment did not induce IFN-α in spleens of IDO-deficient (IDO-KO) mice (Fig. 1A, upper panels). Failure to induce IFN-α was not attributable to defective CD19+ DC development, or to a lack of signaling mechanisms needed to induce IFN-α, because TLR9 ligation induced IFN-α expression comparably in both IDO-sufficient (IDO-WT) and IDO-KO mice (Fig. 1A, lower panels). These findings suggested that an intact IDO gene was essential to induce IFN-α production following B7 ligation, but not following TLR9 ligation.

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Figure 1. IDO controls IFN-α expression after B7 ligation. (A) IDO-WT and IDO-KO mice were treated with CTLA4-Ig or CpG-ODN for 5 h, and spleen sections were stained with anti-IFN-α mAb. (B) RNA samples from untreated DC (–), DC cultured with 100 µg/mL CTLA4-Ig for 5 h (+), or DC infected with influenza virus (flu) were analyzed by RT-PCR to detect IFN-α and β-actin transcripts. (C) Mo-Flo-sorted DC subsets from IDO-WT or IDO-KO mice were treated with CTLA4-Ig for 5 h. IFN-α in supernatants was measured by ELISA. Data shown are representative of at least three experiments.

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To further investigate this finding, we assessed IFN-α gene transcription and IFN-α secretion after CTLA4-Ig treatment (Fig. 1B, C). As reported previously, B7 ligation induced transcription of the IFNα1–9 gene in splenic DC 19. However, IFNα1–9 transcripts were absent in RNA samples from DC-enriched cell preparations from IDO-KO mice after 5 h of culture with CTLA4-Ig (Fig. 1B). DC from IDO-KO mice had no inherent developmental or functional defects in IFN-α expression because influenza (flu) virus infection induced IFNα1–9 transcription in DC from IDO-WT and IDO-KO mice (Fig. 1B). Consistent with data from RNA analyses, sorted DC from IDO-KO mice secreted no detectable IFN-α following CTLA4-Ig treatment (Fig. 1C). As reported previously 19, sorted CD19+ DC from IDO-WT mice were the only splenic cells to express IFN-α after CTLA4-Ig treatment; this response occurred rapidly within 1 h of ligating B7 (data not shown). CD19+ DC consistently produced more IFN-α (on a per cell basis) than other sorted DC (CD11c+ CD19neg, CD11clow), which produced significantly lower or undetectable amounts of IFN-α, respectively, after B7 ligation (Fig. 1C). Thus, CD19+ DC were hyper-responsive to B7 ligation. Collectively, these data showed that CD19+ DC from IDO-KO mice exhibited a specific defect in response to B7 ligation, suggesting that IDO was an essential component of signaling mechanisms leading to IFN-α expression by CD19+ DC.

Pharmacologic inhibition of IDO blocks IFN-α production following B7 ligation

As an alternative approach to test if IDO enzyme activity was essential for IFN-α production following B7 ligation, we treated DC with CTLA4-Ig in the presence or absence of the specific IDO inhibitor, 1-methyl-D-tryptophan (1mT), and measured IFN-α secretion 5 h later (Fig. 2). DC from IDO-WT mice secreted no detectable IFN-α following B7 ligation in the presence of 1mT (Fig. 2A) at concentrations above 0.25 µM (Fig. 2B). Addition of excess tryptophan (to 250 µM, 10× normal) also blocked IFN-α secretion by IDO-WT DC following B7 ligation (Fig. 2A). As before, IDO-KO DC failed to secrete IFN-α following B7 ligation in every treatment group. These data suggested that the key biochemical change that triggered IFN-α expression after B7 ligation was IDO-mediated depletion of free tryptophan.

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Figure 2. IDO enzyme activity is essential for IFN-α production following B7 ligation. (A) AutoMACS-enriched (CD11c+) DC from IDO-WT and IDO-KO mice were treated with 100 µg/mL CTLA4-Ig for 5 h in the presence or absence of 1mT or 10× tryptophan. Control DC were treated with 100 µg/mL mCTLA4-Ig. IFN-α levels in supernatants were measured by ELISA. *p <0.001. (B) IFN-α production by IDO-WT DC treated with 100 µg/mL CTLA4-Ig in the presence of varying concentrations of 1mT (0–25 µM) were measured by ELISA. *p <0.001. (C) AutoMACS-enriched (CD11c+) IDO-WT (black bars) and IDO-KO (white bars) DC were treated with 6.25 µg/mL CpG-ODN or non-CpG-ODN in the presence or absence of 1mT for 5 h. IFN-α levels in supernatants were measured by ELISA. *p <0.05. (D) IFN-α production by Mo-Flo-sorted CD11chigh (Hi) and CD11clow (Lo) DC after CpG-ODN treatment as in (C). Data shown are representative of at least three experiments.

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In contrast, IFN-α production triggered by CpG-ODN was not dependent on IDO enzyme activity (Fig. 2C). After TLR9 ligation, IDO-WT and IDO-KO DC produced similar levels of IFN-α, and IFN-α production by DC from IDO-WT and IDO-KO mice in response to CpG-ODN was reduced by 1mT (∼30%), but not completely abrogated (Fig. 2C), as occurred after B7 ligation. Reduced IFN-α production after TLR9 ligation may be caused by IDO-independent effects of 1mT on DC, as has recently been reported by Agaugue et al.22. As expected, ODN containing no CpG motifs (non-CpG) did not stimulate IFN-α production by DC. To evaluate which DC subsets produced IFN-α after TLR9 ligation, we sorted CD11chigh and CD11clow (includes most conventional splenic pDC) subsets using preparative flow cytometry and analyzed IFN-α secretion by sorted DC after 5 h of incubation with CpG-ODN (Fig. 2D). Although both DC subsets produced IFN-α, sorted CD11chigh DC produced considerably more IFN-α than sorted CD11clow DC.

DC from GCN2-kinase-deficient mice fail to produce IFN-α following B7 ligation

In a recent study, we reported that IDO activity in CD19+ DC prevented proliferation of activated T cells by stimulating general control of non-derepressible-2 (GCN2)-kinase activity in T cells 7. GCN2-kinase senses increased concentrations of uncharged tRNA due to reduced access to free amino acids, and is a component of the cellular integrated stress response (ISR) triggered by amino acid withdrawal 23. By analogy, we hypothesized that cell-autonomous activation of the ISR pathway in CD19+ DC themselves might enhance IFN-α production in response to B7 ligation. To test this hypothesis, we treated GCN2-kinase-deficient (GCN2-KO) mice with CTLA4-Ig and stained spleen sections to detect cells expressing IFN-α after 5 h (Fig. 3A). No IFN-α+ cells were detected in spleen sections from GCN2-KO mice. As expected, IFN-α+ cells were present and dispersed throughout the red pulp areas of the spleen in control, background-matched (129/Sv) GCN2-kinase-sufficient mice. This outcome suggested that an intact GCN2-kinase gene was essential for IFN-α production by CD19+ DC following B7 ligation. Furthermore, this finding was consistent with the notion that GCN2-kinase activation was stimulated by reduced access to free tryptophan caused by IDO activity.

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Figure 3. IFN-α production by DC is GCN2-kinase-dependent following B7 ligation, but not after TLR9 ligation. (A) 129/Sv and GCN2-KO mice were treated with 100 µg CTLA4-Ig. After 5 h, mice were sacrificed and spleen sections were stained to detect IFN-α. (B) AutoMACS-enriched DC from the mice indicated were cultured with 100 µg/mL CTLA4-IgG2a (+) or IgG2a (Ig); no RNA was added to parallel reactions (–). After 5 h, RNA was extracted and IDO and β-actin transcripts were detected by RT-PCR. (C) DC were treated with CpG-ODN (+) for 5 h, and IDO and IFN-α transcripts were detected by RT-PCR. (D) DC from 129/Sv (filled bars) or GCN2-KO (open bars) mice were cultured with non-CpG-ODN or CpG-ODN for 5 h and IFN-α secretion was measured by ELISA. Data shown are representative of at least three experiments.

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In previous studies, we reported that IDO protein was first detected ∼18 h after B7 ligation, well after IFN-α production began 1–5 h after exposure to B7 ligands 18, 19. However, the methods we used to detect IDO protein were relatively insensitive, and only small amounts of pre-formed IDO enzyme (or enzyme synthesized rapidly after B7 ligation) may be sufficient to stimulate an early GCN2-kinase-dependent stress response in CD19+ DC. To test if IDO transcription was induced rapidly after B7 ligation, we analyzed RNA samples from DC cultured for 5 h with CTLA4-Ig (Fig. 3B). IDO transcripts were present in RNA samples from splenic IDO-WT DC treated with CTLA4-Ig, but treatment with control IgG2a antibody did not induce early IDO transcription. Thus, IDO transcription occurred within a few hours of B7 ligation, well before high-level IDO protein expression and IDO enzyme activity was detectable using less sensitive methods in CD19+ DC, and before CD19+ DC acquired potent IDO-dependent T cell-suppressive functions. CTLA4-Ig treatment also induced rapid IDO transcription in DC from GCN2-KO mice (Fig. 3B), showing that signaling mechanisms linking B7 ligation with induction of IDO transcription were intact in DC from GCN2-KO mice. As expected, no IDO transcripts were detected in DC from IDO-KO mice.

To test if GCN2-kinase ablation affected TLR9 signaling in pDC, we analyzed IDO and IFN-α expression in RNA samples from DC from GCN2-KO and control 129/Sv mice exposed to CpG-ODN (Fig. 3C, D). As expected, IFN-α expression following TLR9 ligation was not affected by genetic ablation of GCN2-kinase. Although IFN-α production was not dependent on IDO expression, early IDO transcription was also induced by CpG-ODN exposure in DC from GCN2-KO and 129/Sv control mice.

IDO-competent CD19+ DC develop normally in IDO- and GCN2-kinase-deficient mice

To further evaluate if the failure to induce IFN-α in GCN2-KO and IDO-KO mice was due to abnormal development of CD19+ DC in these mice, we performed three-color FACS analyses focused on the small subset of CD11chigh CD19+ DC that were the only splenic DC subset that expressed IFN-α, and subsequently acquired potent IDO-dependent T cell suppressive functions following B7 ligation 19, 20. Using the gating criteria shown in Fig. 4 (see dot plot), splenic CD11chigh CD19+ DC were present in comparable numbers in IDO-KO (3–4% of total DC) and GCN2-KO (6–7% of total DC) mice, relative to background-matched mice (F1[CBA × B6] and 129/Sv, respectively) (data not shown). The higher proportions of IDO-competent CD19+ DC detected in mice with 129/Sv background are consistent with previous reports that 129/Sv mice harbor higher proportions of pDC than other mouse strains 24.

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Figure 4. IDO-competent CD19+ DC develop normally in IDO-KO and GCN2-KO mice. Splenocytes from IDO-KO, GCN2-KO, and background-matched F1[CBA × B6], 129/Sv mice (respectively) were stained with anti-CD11c, -CD19, and -CD80 (B7.1) or -CCR6 mAb, and analyzed on a BD FacsCanto flow cytometer. (A) Histograms show anti-B7.1 and -CCR6 mAb staining profiles for CD11chigh CD19+ cells gated as indicated on the dot plot; markers were set to exclude cells stained with irrelevant isotype-matched mAb (left panels). Proportions of gated CD11chigh CD19+ DC (of total CD11c+ DC) were 3–4% for IDO-KO and F1[CBA × B6] (F1) mice, and 6–7% for GCN2-KO mice and 129/Sv mice (not shown). Percentages indicate the proportion of gated CD19+ DC stained with anti-B7.1 or -CCR6 mAb relative to isotype control mAb (markers were set to 3%); numeric values show mean fluorescence intensities on gated (total) CD19+ DC. (B) Histograms show isotype (left panels) and CCR6 (right panels) staining profiles for other gated DC subsets from F1[CBA × B6] mice, as indicated. Data shown are representative of at least three experiments.

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Another potential reason why CD19+ DC from IDO-KO and GCN2-KO mice might have failed to produce IFN-α following CTLA4-Ig treatment is defective expression of B7 molecules. To address this issue, we stained DC with anti-CD11c, -CD19 and -CD80 (B7.1) mAb. CD19+ DC from IDO-KO and background-matched F1[CBA × B6] mice expressed B7.1 at uniformly high and comparable levels (Fig. 4A), as did DC from GCN2-KO mice and background-matched 129/Sv mice. Similar results were also obtained when DC were stained with CD86 (B7.2) mAb (data not shown). CD19+ DC also expressed uniformly high levels of the CC chemokine receptor CCR6 (Fig. 4A), which is an excellent marker for IDO+ pDC isolated from tumor-draining lymph nodes of mice bearing B16 melanomas 25, as well as IDO+ pDC prepared from human PBMC 14. Indeed, CD19+ DC expressed higher levels of CCR6 than CD11chigh CD19neg (mostly myeloid) and CD11clow CD19neg (mostly plasmacytoid) DC (Fig. 4B), emphasizing the unique phenotypic characteristics of the CD19+ DC subset of pDC competent to express IFN-α and IDO. Thus, the only defect we detected in DC from GCN2-KO and IDO-KO mice was failure of CD19+ DC to express IFN-α following B7 ligation, even though CD19+ DC were present in normal numbers and expressed normal levels of B7 and CCR6. This functional defect was highly specific to signaling via B7 because the ability of DC to respond to TLR9 ligation was not abrogated in IDO-KO and GCN2-KO mice, or by pharmacologic inhibition of IDO. Collectively, these data are consistent with the hypothesis that IDO-GCN2 signaling is a necessary component of B7-mediated induction of IFN-α expression in CD19+ DC.

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements

In this report, we identify an unanticipated and novel role for IDO as an essential upstream triggering mechanism to induce IFN-α production by CD19+ DC following B7 ligation. In previous studies, we and others identified specific subsets of murine splenic DC that could be induced to express IDO using several ligands. As a consequence, DC acquired potent IDO-dependent T cell regulatory functions that blocked effector T cell responses and promoted differentiation of CD4+CD25+ regulatory T cells (Treg cells) 2, 13, 15, 19, 20, 2628. Here, we show that IDO expressed rapidly after B7 ligation triggered IFN-α expression by CD19+ DC, most likely due to activation of the GCN2-kinase-dependent stress response to tryptophan withdrawal. Thus, following B7 ligation, IDO enzyme activity is required both upstream and downstream of IFN-α expression to convert CD19+ DC into DC with potent T cell regulatory functions.

Signaling mechanisms involving positive feedback loops to amplify initial responses are not unusual in biological control of immune effector functions. For example, IFNAR signaling amplifies downstream IFN-α production in some cell types (including some DC). Following viral infection, pDC are predisposed to produce IFN-α via IRF7 signaling due, in part, to IRF7 expression 2932. Moreover, the Th2 cytokine IL-4 promotes Th2 differentiation leading to amplified IL-4 production 33. The biological significance of positive feedback loops that amplify immune effector mechanisms may be to ensure that key checkpoints are available to help prevent inappropriate or premature commitment to specific differentiation pathways that could profoundly impact subsequent immune outcomes. Hence, it may be critically important to regulate IDO expression in DC because they acquire potent and dominant T cell-suppressive functions due to direct regulation of activated effector T cells, and stimulation of Treg cell differentiation, which mediate bystander suppression. Nevertheless, it is currently unclear why IDO up-regulation following B7 ligation is subject to more stringent control than IDO up-regulation following TLR9 ligation.

The ability of CD19+ DC to express IFN-α after TLR9 ligation was unaffected by pharmacologic or genetic ablation of IDO, or by genetic ablation of GCN2-kinase. In a series of recent reports, Honda and colleagues described that formation of stable endosomal complexes containing MyD88 and IRF7 was essential to induce IFN-α expression in murine pDC following TLR9 ligation 3436. CD19+ DC resemble conventional pDC as they display plasmacytoid morphology, express B220, and produce IFN-α following TLR9 ligation 15, 1820. Thus, TLR9 ligation may induce IFN-α expression in CD19+ DC via the MyD88/IRF7-dependent pathway described for conventional pDC. However, CD19+ DC are distinct from conventional pDC because they do not express 120G8, can be induced to express IDO, and are located exclusively in the splenic red pulp. Thus, the obligatory requirement for early (upstream) IDO activity to trigger IFN-α expression after B7 ligation may be unique to CD19+ DC. Nevertheless, it is unclear how the obligatory requirement for induction of an early IDO-GCN2 stress response might trigger IFN-α production in CD19+ DC. One possibility is that IDO-GCN2 signaling facilitates formation of stable MyD88-IRF7 endosomal complexes in CD19+ DC, perhaps as a consequence of activating the downstream stress response (ISR) pathway. TLR9 ligands have multiple effects on DC functions other than inducing IFN-α expression. Hence, requirements for upstream IDO-GCN2 signaling following B7 ligation may be circumvented by simultaneous activation of other mechanisms after TLR9 ligation.

DC expressing IDO stimulate GCN2-kinase-dependent stress responses in T cells that promote suppressive outcomes. Effector T cells from GCN2-KO mice were insensitive to the suppressive functions of IDO+ DC, and naive CD4+ T cells from GCN2-KO mice displayed a partial defect in IDO-mediated enhancement of Treg cell differentiation 7, 28. By analogy, we hypothesize that IDO activity in CD19+ DC also stimulated a cell-autonomous GCN2-kinase-dependent stress response in DC themselves, which then triggered IFN-α expression by as yet undefined downstream signaling mechanisms. GCN2-kinase senses increased uncharged tRNA at ribosomal tRNA binding sites and, once activated, GCN2-kinase stimulates a cascade of downstream signaling events that culminate in profound changes in gene expression and cellular functions, including down-regulation of protein synthesis 23. Thus, induction of the GCN2-kinase-dependent stress response may have wide-ranging effects on the cell biology and functions of CD19+ DC.

It is unclear how CTLA4-Ig stimulates early IDO enzyme activity in CD19+ DC. Previous reports showed that IFN-γ and IFN-α can independently induce IDO in DC, though specific requirements for IFN-γ versus IFN-α signaling depended on several factors, including the nature of the ligand used to induce IDO, the subset of DC under scrutiny, and the methods used to assess functional IDO expression 13, 1820, 27. These findings are consistent with earlier reports that IFN types I and II both possess IDO-inducing activity 10, and with the finding that IDO gene promoters in humans and mice contain IFN-stimulatory response elements (ISRE; IFN-α responsive) and gamma-activating sequence (GAS; IFN-γ responsive) motifs that stimulate gene transcription when bound to transcriptional complexes containing activated STAT proteins and other regulatory proteins 11, 12. As we have reported previously, genetic ablation and antibody blockade of IFN-γ signaling had no effect on IDO up-regulation in CD19+ DC, or on their ability to suppress T cell responses in an IDO-dependent fashion following CTLA4-Ig treatment 18, 19. However, both genetic ablation and antibody blockade of IFN-α signaling prevented IDO up-regulation in CD19+ DC, which retained potent T cell stimulatory functions as a consequence. IFNAR signaling was also indispensable for IDO up-regulation in CD19+ DC after TLR9 ligation, while blockade of IFN-γ receptor signaling had no effect on IDO induction in CD19+ DC after CpG-ODN treatment 20. Thus, IFN-α expression is essential to amplify IDO expression in CD19+ DC, and to effect a critical change in DC functions from T cell-stimulatory to T cell-suppressive functions. Irrespective of whether IFN-α or IFN-γ is responsible for amplifying IDO expression in CD19+ DC, it is currently unclear how B7 ligation induces IDO prior to IFN-α expression. Signals generated as a consequence of ligating surface B7 molecules on CD19+ DC may activate pre-formed, inactive IDO protein to stimulate enzyme activity, or induce de novo synthesis of enzymatically active IDO needed to trigger GCN2-kinase. With regard to these two possibilities, it may be important that IDO transcription in DC was induced rapidly after B7 and TLR9 ligation in the present study.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements

Mice

F1[CBA × B6], IDO-KO, and GCN2-KO mice were bred in a specific pathogen-free facility; 129/Sv mice were purchased (Taconic). IDO-KO and GCN2-KO mice have been described 7, 15, 37. All procedures involving mice were approved by the Institutional Animal Care and Use Committee.

CTLA4-Ig

Cytolytic (Cat. No. C4483; Sigma, St Louis, MO) and non-cytolytic (Cat. No. MF120A4; Chimerigen, Boston, MA) CTLA4-IgG2a and IgG2a (Cat. No. 14–4724–85; BD Biosciences, San Diego, CA) reagents were injected into mice (100 µg, intraperitoneally) and DC were cultured with 100 µg/mL CTLA4-Ig or IgG2a.

CpG-ODN

CpG-ODN (1826) and non-CpG-ODN (2138) were purchased from Coley Pharmaceutical Group, Inc. (Ontario, Canada). To induce IDO, mice were injected with 50 µg CpG-ODN intravenously, as described 20. DC were cultured with 6.25 µg/mL ODN.

1mT and 10× tryptophan

1mT (Cat. No. 110117–83-4) and L-tryptophan (L-Trp, Cat. No. 73–22-3) were purchased from Sigma-Aldrich (Milwaukee, WI) and added to final concentrations of 100 µM and 245 µM, respectively, unless otherwise stated.

Antibodies

Rat monoclonal (Cat. No. 22100-1) and rabbit polyclonal (Cat. No. 32100-1) anti-murine antibodies against IFN-α were purchased from PBL Biomedical Laboratories (Piscataway, NJ).

Splenic DC isolation

Spleens were harvested and DC were enriched by AutoMACS or sorted into subpopulations by Mo-Flo as described previously 19. DC subset sorting was performed in the MCG Flow Cytometry Core Facility using a Mo-Flo four-way flow cytometer (Dako Cytomation) equipped with 488 nm argon (for FITC, PE, PE-Cy5) and 647 nm krypton (for allophycocyanin) lasers. Cells were gated based on forward and side scatter properties and on marker combinations to select cells of interest.

Immunohistochemistry

For IFN-α detection, spleens were collected from mice 5 h after treatment and fixed in 10% formalin. Preparation of tissue sections and IFN-α staining have been described 19. Tissue sections were counterstained with hematoxylin (Richard-Allan Scientific, Kalamazoo, MI) and mounted in Faramount aqueous mounting media (S3025; DAKO).

RT-PCR

IFN-α and IDO expression were analyzed by semi-quantitative RT-PCR. Procedures for RNA isolation, primers for IFN-α subtypes, and RT-PCR conditions have been described 19. Infection with influenza A virus strain X31 (courtesy of Dr. Graeme Price) has been described 19. Primers were designed to amplify specific DNA products from IDO mRNA (5′-AGACC ACCAC ATAGA TGAAG-3′ and 5′-CCACC AATAG AGAGA CGAGG A-3′).

ELISA

CD11c+ AutoMACS-enriched cells (106) were cultured with 100 µg/mL CTLA4-Ig or 6.25 µg/mL ODN. After 5 h, culture media were harvested and IFN-α concentration was measured as per the manufacturer's instructions (Mouse IFN Alpha ELISA kit; PBL Biomedical Laboratories). The sensitivity limit of the assay is 10 pg/mL.

Flow cytometry

Splenocytes were prepared following collagenase digestion (400 U/mL; 30 min at 37°C) and RBC lysis (ACK lysis buffer), incubated with Fc receptor block (BD Pharmingen; Cat. No. 553142, clone FcγIII/II 2.4G2) prior to incubation with dye-conjugated antibodies to CD11c-[allophycocyanin] (BD Pharmingen; Cat. No. 550261, clone HL3), B220-[PerCP] (BD Pharmingen; Cat. No. 553093, clone RAB-632), CD19–[FITC] (BD Pharmingen; Cat. No. 557398, clone 1D3), CD80-[FITC] (BD-Pharmingen: Cat. No. 553768, clone 16–10A1), CCR6-[PE] (R&D Systems, Cat. No. FAB195P, clone 53103) for 30 min on ice. Dye-conjugated isotype control mAb were used to detect non-specific staining and autofluorescence. Flow cytometry was performed on a BD FacsCanto flow cytometer equipped with a blue (488 nm, air-cooled, 20 mW solid state) and red (633 nm, 17 mW HeNe) excitation system. Data analysis was performed using FACSDiva software.

Statistics

Data in experimental and control groups were analyzed by two-sample t-test. p-Values less than 0.05 are considered significant. NCSS software was used for statistical analyses.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements

We thank Jeanene Pihkala (manager, MCG Flow Cytometry Core facility) for expert assistance with Mo-Flo sorting and Anita Wylds, Doris McCool, and Erika Thompson for expert technical assistance. This work was supported by NIH grants to A.L.M. (HD41187, AI063402) and D.H.M. (CA103320, CA096651). D.H.M. and A.L.M. have intellectual property interests in the therapeutic use of IDO and IDO inhibitors, and receive consulting income from NewLink Genetics, Inc., which holds a license to the technology.

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