| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
* Institute of Molecular Medicine and Genetics and Departments of
Pediatrics and
Medicine, Medical College of Georgia, Augusta, GA 30912
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
One mechanism by which APCs may regulate T cell responses is through the expression of indoleamine 2,3-dioxygenase (IDO),3an enzyme that degrades the essential amino acid tryptophan ( 5). We and others have shown that the expression of IDO by human monocyte-derived macrophages ( 6) and DCs (7, 8) allows these APCs to inhibit T cell proliferation in vitro. Transfection of recombinant IDO into non-APC cell lines confers the ability to inhibit Ag-specific T cell responses ( 9). In vivo, using murine models, a role for IDO has been demonstrated in maternal tolerance toward the allogeneic fetus ( 10, 11), suppression of T cell responses to MHC-mismatched allografts ( 10, 12), control of T cells in autoimmune disorders ( 13, 14, 15), and suppression of immune responses to tumors ( 16, 17).
A mechanistic link has also been demonstrated between the tolerance-inducing activity of recombinant CTLA4-Ig Fc fusion protein (CTLA4-Ig), a synthetic fusion protein that binds with high affinity to the costimulatory molecules B7-1 and B7-2, and the fact that CTLA4-Ig also induces IDO expression in DCs ( 18). Engagement of B7-1/B7-2 molecules on murine DCs by CTLA4-Ig was found to activate a signaling pathway leading to induction of IDO in vitro, and pharmacologic inhibition of IDO in vivo completely abrogated the tolerogenic effect of CTLA4-Ig in a mouse islet cell transplant model ( 18). Recently, the physiologic CTLA4 receptor expressed by murine regulatory T cells (Treg) has been shown to mediate an analogous IDO-inducing effect in vitro ( 19). We have reported that the up-regulation of IDO by CTLA4-Ig in vivo is highly selective, being restricted to the CD8
+ subset of DCs and to B220+ (plasmacytoid) DCs, but despite the limited number of such cells, IDO expression could completely suppress clonal expansion of large cohorts of adoptively transferred alloreactive T cells ( 20). Thus, in a variety of murine models, IDO expression by DCs functions as an inducible immunosuppressive mechanism, which may be triggered at least in part by the Treg system.
In the human system, IDO expression has been observed by immunohistochemistry in placenta, tumor-draining lymph nodes, and primary tumors ( 17, 21), but the functional immunoregulatory role of IDO in humans is less well defined. To model IDO-mediated immune regulation by human DCs we have developed an in vitro culture system in which monocyte-derived DCs can be enriched for a population expressing high levels of IDO ( 7). Using this system we have demonstrated that maturation does not in and of itself abrogate IDO-mediated suppression, allowing some DCs to manifest a phenotype that is both mature and actively suppressive ( 7).
In mature IDO+ DCs the functional activity of IDO is tightly regulated, with the IDO protein being constitutively expressed, but enzymatic activity manifest only in response to specific triggering signals ( 7). The most potent trigger for functional activity was found to be exposure to activated T cells during Ag presentation. We initially hypothesized that this might be due to T cell-derived IFN-
, because IFN- is produced early during T cell activation ( 22, 23), and IFN- from T cells induces IDO in human monocyte-derived macrophages ( 6). However, we found that simply treating isolated DCs with rIFN- did not recapitulate the IDO-inducing effect of activated T cells and, in fact, produced the diametrically opposite effect, i.e., marked down-regulation of IDO protein, mRNA, and enzymatic activity ( 7). Thus, activated T cells supplied an additional signal, over and above IFN-, which was obligately required to trigger functional IDO. Based on observations from the murine system, we hypothesized that this IDO-inducing signal might be ligation of B7 molecules on the DCs by their counter-receptors (CTLA4 and CD28) on T cells.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Human monocytes (>90% purity) and lymphocytes (>80% T cells, balance of B and NK cells, <1% monocytes) were isolated by leukocytophoresis and counterflow elutriation ( 6) under a protocol approved by our institutional review board. To generate DCs, monocytes were cultured for 7 days in serum-free X-vivo15 medium (BioWhittaker, Walkersville, MD) supplemented with GM-CSF (50 ng/ml; R&D Systems, Minneapolis, MN) and IL-4 (50 ng/ml; R&D Systems) on days 0, 2, and 4 as previously described ( 7). Unless otherwise specified, all DCs were matured by adding the following mixture ( 24) during the final 48 h of culture: TNF- (1100 U/ml; BD PharMingen, San Diego, CA), IL-1
(1870 U/ml; BD PharMingen), IL-6 (1000 U/ml; BD PharMingen), and PGE2 (1 µg/ml; Sigma-Aldrich, St. Louis, MO). Under these conditions, IDO+ cells constituted up to 60–70% of all DCs (in contrast to the DCs generated in bovine serum-based medium, which typically contained only 20–30% IDO+ DCs). The nonadherent (IDO+) fraction was harvested by gentle aspiration (without vigorous pipetting) and used in all assays. Optimal fractionation of IDO+ cells was obtained at intermediate (rather than high) levels of GMCSF, and good results were also obtained with a single initial feeding of GM-CSF without further supplementation.
The adherent cells from these cultures were CD14+ and appeared to represent a transitional or macrophage-like fraction, as previously described ( 7). They did not show significant IDO-mediated suppression and were not used in this study. In our system, the distinction between adherent and nonadherent fractions did not represent a unique, fixed lineage commitment, because the relative proportions of the two populations could be influenced by the culture conditions (and, for example, in bovine serum-based cultures, both IDO+ and the IDOneg cells were nonadherent ( 7)). However, the conditions used in this study generated a reliably enriched population of IDO-expressing DCs, which could be readily recovered in the nonadherent fraction.
For isolated T cell subsets, elutriator-purified lymphocytes were incubated with either anti-CD4 (clone T-310; DAKO, Carpinteria, CA) or anti-CD8 Ab (clone DK25, DAKO) for 30 min on ice, washed, and isolated by immunomagnetic bead sorting (Miltenyi Biotec, Auburn CA). The purity of each fraction was 90–95%. Normally, to avoid artifacts introduced by staining and sorting, elutriator-purified lymphocytes were used without fractionation, unless CD4 and CD8 subsets were specifically required. However, direct head-to-head comparison of unfractionated lymphocytes vs sorted/remixed (CD4 and CD8) T cells from the same apheresis showed similar patterns of response (as shown in Results), so the sorting process did not appear to adversely affect the T cells.
V-bottom MLR system
For MLRs, nonadherent DCs were mixed with 5 x 105 allogeneic lymphocytes (unfractionated) or with the number of sorted CD4 or CD8 cells that would have been present in the original 5 x 105 cells. Unless otherwise specified, an APC:T cell ratio of 1:20 is shown in all figures. (In the figures, data are shown at a single APC:T cell ratio for simplicity, but comparable results were obtained at various APC:T cell ratios.)
MLRs were performed for 5 days in 250 µl of complete medium (10% FCS in RPMI 1640). Alternatively, where indicated, MLRs were performed in a chemically defined medium comprising tryptophan-free RPMI 1640 supplemented with BSA, insulin, transferrin, and cholesterol, as previously described ( 6). At the beginning of MLRs, replicate groups of five wells received no additive, 200 µM 1-methyl-D-tryptophan (1MT; Sigma-Aldrich), or 250 µM L-tryptophan (Sigma-Aldrich). (To prepare 1MT, a 100x stock solution (20 mM) was dissolved in 0.1 N NaOH, then adjusted to pH 7.4.) After 5 days of MLR, proliferation was measured by 4-h thymidine incorporation assay. In all MLRs, V-bottom culture wells (Nalge-Nunc, Rochester, NY) were used, as previously described ( 7).
For mitogen-activated T cell proliferation, purified lymphocytes were activated with immobilized anti-CD3 Ab plus soluble anti-CD28 (clone CD28.2, as used above), as previously described ( 6).
Blocking Abs
Neutralizing mAb against human IFN- (clone B-B1) was purchased from BioSource International (Camarillo, CA) and used at a supersaturating concentration of 100 µg/ml. Blocking anti-CTLA4 mAb (clone BNI3; BD PharMingen) was used at a concentration of 1 µg/ml. This clone has been used to block CTLA4 function on human T cells in vitro ( 25). Results were confirmed with a second clone against a different epitope (clone 8H5; Ancell, Bayport, MN). Anti-CD28 mAb (clone CD28.2; BD PharMingen; 1 µg/ml) was selected because it both blocks the CD28-B7 interaction (as shown below) and provides costimulation to T cells via CD28 cross-linking. Most intact dimeric anti-CD28 Abs provide costimulation ( 26), and this was empirically confirmed for this clone using purified T cells activated with immobilized anti-CD3 Ab ( 6). Results with this mAb were confirmed with a second clone (5D10; Ancell). Abs were added at the start of MLRs. The strategy of blocking CTLA4/CD28 (rather than blocking B7-1/B7-2) was chosen because Abs against B7-1/B7-2 might cross-link their targets, thus inadvertently generating the signal we wanted to block ( 27). In preliminary validation experiments, none of the blocking Abs used had any effect when T cell proliferation was stimulated by fresh monocytes plus anti-CD3 mAb (a system in which there is no IDO-mediated suppression of T cells).
To confirm that the anti-CD28 and anti-CTLA4 Abs were blocking Abs, we measured their ability to inhibit binding of soluble recombinant CD28-Ig Fc fusion protein (CD28-Ig) and CTLA4-Ig fusion proteins to DCs. DCs were incubated for 30 min on ice with either biotinylated CD28-Ig (Ancell) or CTLA4-Ig (Chimerigen, Allston, MA), both at 10 µg/ml. All incubations and washes contained 0.02% sodium azide to reduce capping. Duplicate tubes received either the blocking Ab being tested or an equivalent amount of isotype-matched mouse IgG. Cells were washed and fixed, and the bound ligand was quantitated by FACS using PE-conjugated anti-human IgG detection reagent (for CTLA4-Ig) or streptavidin-PE (for CD28-Ig).
Anti-CD80 mAb (clone L307.4) and anti-CD86 mAb (clone IT2.2; both from BD PharMingen; used at 1 µg/ml) have been shown to generate intracellular signals in B cells when cross-linked ( 28, 29). In preliminary validation experiments we found that isolated DCs did not require any additional cross-linking of these Abs to show a biologic effect (because DCs themselves are potent cross-linkers of Abs, as demonstrated by their ability to cross-link anti-CD3 Abs and activate T cells). However, in MLRs, where DCs were present at much lower densities, we found that a cross-linking reagent was required for the full effect. This cross-linking could be provided either by F(ab')2 of goat anti-mouse Ab (1 µg/ml; Jackson ImmunoResearch, West Grove, PA) or by biotin-avidin cross-linking (60 ng/ml streptavidin with biotinylated primary Ab); both strategies produced similar results. The CD80/CD86 Abs were optimally effective when added on day 2 of MLR, just before the onset of T cell proliferation.
HPLC measurement of kynurenine
Culture supernatants (150 µl) were extracted with 1.3 ml of methanol, the precipitate was removed by centrifugation, and the supernatant was dried under vacuum. Samples were resuspended in an initial mobile phase (deionized water), injected onto a C18 column (Luna C-18; 250 x 4.6 mm, 5 µm; Phenomenex, Torrance, CA), and eluted with a linear gradient of acetonitrile in water (0–80% over 20 min). Absorbance was measured at 225 nm and compared against standard curves for tryptophan, kynurenine, and 1MT. The limit of detection for this assay was 1.2 µM.
Flow cytometry
Phenotyping Abs and reagents were obtained from BD PharMingen unless specified. Nonadherent DCs and adherent APCs were stained with anti-CD123-biotin (clone 7G3), followed by streptavidin-PerCP, and anti-CD11c-allophycocyanin (clone S-HCL-3). After fixation and permeabilization (Cytofix/Cytoperm), cells were stained with affinity-purified rabbit Ab against human IDO (generated as previously described ( 7)), followed by R-PE-labeled anti-rabbit secondary Ab (Jackson ImmunoResearch Laboratories; cross-adsorbed against mouse, human, and bovine IgG). For all experiments, the negative control for IDO staining was the anti-IDO Ab preadsorbed with a 50-fold molar excess of the immunizing peptide (DLIESGQLRERVEKLNMLC). Dendritic cells were gated on forward and side scatter. For FACS on MLRs, the cocultures were harvested after 48 h, treated for 5 min with EDTA to disrupt clusters, then stained as described. For analysis of T cells, B cells were excluded from the lymphocyte gate by CD20 labeling.
Immunohistochemistry
Cytocentrifuge preparations were fixed for 10 min in 10% formalin, then primary Abs were applied: either rabbit anti-human IDO (0.05 µg/ml) or anti-CTLA4 (clone BNI3; 1 µg/ml). IDO was detected with rabbit-specific secondary Ab conjugated to Alexa-488 (Molecular Probes, Eugene, OR) cross-adsorbed against mouse IgG; CTLA4 was detected with anti-mouse Ab conjugated to Alexa-568, cross-adsorbed against rabbit IgG (Molecular Probes). Cells were harvested from MLRs with minimal manipulation to preserve T cell-DC conjugates for confocal analysis ( 30).
Statistical analysis
Multiple treatment groups were compared within individual experiments by ANOVA. All experiments were repeated at least three times unless more replicates are specified.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The culture system for monocyte-derived DCs was optimized specifically to generate suppressive DCs and differed somewhat from other systems. Relevant variables included the use of a highly purified starting population of monocytes, the use of X-vivo15 medium without serum supplementation, maturation with a TNF-based cytokine mixture, and harvesting only the nonadherent cell fraction, all as described in Materials and Methods. Fig. 1a shows that the nonadherent cells from these cultures were preferentially enriched for DCs expressing IDO compared with the adherent cell fraction from the same cultures. The nonadherent cells (hereafter IDO+ DCs) had the phenotype previously described ( 7), including coexpression of CD11c and CCR6 and low, but consistent, levels of CD123 (IL3-R; Fig. 1a). These markers were present before maturation and were unchanged after maturation. After maturation, the IDO+ DCs were >80% CD83+ and expressed high levels of HLA-DR, CD80, and CD86, consistent with a mature phenotype ( 7).
|
Using the preceding culture system and MLR assay, we compared the activities of immature vs mature IDO+ DCs (Fig. 1c). In the absence of 1MT, neither immature nor mature DCs were good stimulators of T cell proliferation. However, when IDO activity was blocked with 1MT, the mature DCs were revealed as excellent stimulators of T cell responses (Fig. 1c) despite their potent suppression when IDO was active. Identical results were obtained when the tryptophan-depleting effect of IDO was bypassed by adding excess tryptophan to the medium. Neither 1MT nor 10x tryptophan had any effect on T cell proliferation when the T cells were activated using immobilized anti-CD3/CD28 mAbs (data not shown), confirming that these agents did not have any intrinsic, nonspecific stimulatory activity for T cells.
Ligation of B7-1/B7-2 prevents down-regulation of IDO in isolated DCs
Mature IDO+ DCs activated in isolation (using recombinant IFN-, but without T cells) markedly down-regulated IDO (Fig. 2a). However, this down-regulation could be prevented if a second signal was supplied through ligation of B7 molecules (CD80 or CD86). Fig. 2a (panels 3 and 4) show that Ab-mediated cross-linking of CD80 or CD86 fully rescued the isolated DCs from IFN--induced down-regulation of IDO. The FACS data were confirmed by analysis of IDO enzymatic activity (Fig. 2b), measured as accumulation of kynurenine in the culture supernatants. (Kynurenine is the first stable downstream metabolite produced by IDO. Although it is not a quantitative measure of enzyme activity, because its accumulation is influenced by the degree of conversion to subsequent downstream metabolites ( 32), it is a highly specific qualitative marker for IDO activity.) By itself, IFN- produced a small and transient increase in functional activity (the figure reflects the amount of kynurenine accumulating over the first 24 h), but after 24 h, the expression of IDO protein had been lost (see Fig. 2a), and IDO enzymatic activity returned to background levels. Likewise, CD80 or CD86 engagement alone produced a variable, but always minor, amount of IDO activity. However, IFN- and B7 engagement together acted synergistically, allowing IDO protein expression to be maintained (Fig. 2a) and achieving full enzymatic activity (Fig. 2b).
|
in the induction of IDO. Fig. 2c shows that CD28-Ig was markedly less effective on an equimolar basis than CTLA4-Ig at inducing IDO. Even when CD28-Ig was extensively multimerized by avidin-biotin cross-linking, it was still less effective than CTLA4-Ig at inducing IDO. B7-1/B7-2 and their corresponding counterreceptors are expressed during MLR
The preceding experiments used only isolated DCs. We therefore asked which of the preceding molecules were actually present during MLRs. Fig. 3a shows that the DCs expressed both CD80 and CD86, and that the majority of activated T cells (identified by CD25 expression) expressed CD28 (Fig. 3b). Consistent with previous reports ( 33), we were unable to detect CTLA4 on the surface of most T cells by FACS analysis (data not shown), but we readily detected intense, localized accumulations of intracellular CTLA4 by fluorescence microscopy in a small subset of activated T cells (Fig. 3, c and d). Thus, B7-1, B7-2, CTLA4, and CD28 were all expressed in the MLR system.
|
To test the hypothesis that engagement of B7 molecules triggered IDO expression during Ag presentation, we used blocking Abs against the extracellular domains of CTLA4 and CD28. These Abs inhibited binding of soluble CTLA4-Ig and CD28-Ig fusion proteins to IDO+ DCs (Fig. 4a), confirming that the Abs blocked the physiologic interaction of their respective target molecules with B7. To ensure that T cells were not deprived of costimulation, an activating clone of anti-CD28 Ab was used ( 6).
|
The preceding results were confirmed at the level of IDO enzymatic activity (kynurenine accumulation in the MLR supernatants), as shown in Fig. 4c. We found that blocking either CTLA4 or CD28 alone caused a significant decrease in overall kynurenine production (a finding not evident from the less sensitive T cell proliferation readout in Fig. 4b), thus indicating that the roles of CTLA4 and CD28 were not simply redundant. When the two Abs were used together, they abolished all detectable IDO activity, corresponding to the loss of suppressor activity seen in Fig. 4b. (Accumulation of kynurenine in the supernatant is a useful qualitative marker of IDO activity, but the kynurenine itself does not cause T cell inhibition, and the quantitative level of kynurenine does not predict the degree of T cell suppression.)
Finally, we asked whether IDO+ DCs that failed to receive a signal via B7 during MLR would behave like the isolated DCs shown in Fig. 2a, i.e., would down-regulate IDO instead of up-regulating it. Fig. 4d shows that the DCs normally up-regulated IDO protein during MLR, typically by 2- to 3-fold (measured by FACS, gating selectively on the CD11c+CD123+ population to identify the DCs). In contrast, when CTLA4 and CD28 were blocked, IDO was significantly down-regulated during MLR, thus recapitulating the behavior of DCs activated in the absence of T cells.
cross-linking B7-1/B7-2 up-regulates IDO activity in MLR
We next asked whether artificially cross-linking B7-1/B7-2 would restore IDO activity in MLRs when CTLA4 and CD28 were blocked. For these experiments, anti-CD80/CD86 mAbs were used as described in Fig. 2. Fig. 5a shows that Abs against CD80 or CD86 added individually had little effect on IDO. However, simultaneously cross-linking both molecules fully restored inhibition, to a level comparable to that seen when CTLA4 and CD28 were active. This inhibition was reversed by 1MT, indicating that the inhibitory effect of B7-1/B7-2 ligation was due to induction of IDO. Cross-linking CD80/CD86 induced kynurenine production in the same pattern observed for T cell inhibition (Fig. 5b). In additional experiments (not shown), cross-linking B7-1/B7-2 also prevented down-regulation of IDO in MLRs when CTLA4 and CD28 were blocked (i.e., as in Fig. 4d). Thus, by three independent readouts (T cell suppression, kynurenine production, and maintaining IDO expression), ligation of CD80/CD86 restored functional IDO activity in MLRs.
|
IFN- is required for full induction of IDO during MLR
In Fig. 2b we showed that both B7 engagement and IFN- were required to achieve full induction of IDO in isolated DCs. We therefore asked whether endogenously produced IFN- was required for induction of IDO during MLR. Fig. 6a shows that neutralizing Abs against IFN- significantly reduced the expression of IDO enzymatic activity during MLR. As measured by kynurenine production, the effect of neutralizing endogenous IFN- was almost as great as that of blocking CTLA4/CD28. The effect of IFN- neutralization on T cell suppression was not as complete (Fig. 6b), but was still significant. (As shown in Fig. 4c, it is easier to reduce overall kynurenine production than it is to abrogate suppression, suggesting that maximal levels of IDO are not necessary to accomplish suppression.)
|
Many helper/regulatory activities are mediated preferentially by the CD4+ subset of T cells. We asked whether the ability to induce IDO via B7 ligation was associated only with CD4+ T cells or was found in both CD4+ and CD8+ subsets. T cells were fractionated based on CD4 or CD8 expression, as described in Materials and Methods, and each fraction (plus the original unfractionated preparation) was used as responder cells in MLRs (Fig. 7a). All MLRs were stimulated by the same DCs, so any differences in IDO induction were attributable solely to the responder T cells. Fig. 7a shows that isolated CD4+ T cells triggered potent IDO-mediated inhibition (>90% at all time points), which was comparable to the total lymphocyte population without fractionation. In contrast, isolated CD8+ showed excellent proliferation with virtually no 1MT-reversible inhibition. Not only was this pattern different from the isolated CD4+ cells, but it was also markedly different from the behavior of the same CD8+ cells in the unfractionated mixture (where they were fully suppressed). Thus, isolated CD8+ T cells either became refractory to IDO or failed to induce IDO.
|
Tryptophan depletion is required for inhibition of T cell proliferation by IDO
Mechanistically, IDO depletes tryptophan, but it also generates kynurenine and other downstream metabolites. At high concentrations, some of these metabolites can affect T cell proliferation and viability ( 35, 36, 37). To determine whether the effect of IDO in our system was due to depletion of tryptophan or production of toxic metabolites, we added increasing amounts of tryptophan to MLRs, then simultaneously measured the concentration of kynurenine (the first major metabolite) in the medium, and the degree of T cell suppression. As shown in Fig. 8a, adding more tryptophan progressively increased the amount of kynurenine produced, but it also progressively enhanced T cell proliferation (i.e., reversed the IDO-mediated suppression). At the highest concentrations of tryptophan, T cell proliferation reached the same level seen when IDO activity was blocked by 1MT. Thus, suppression was lowest in the groups in which metabolite production was highest, and highest where metabolites were lowest, arguing strongly against a causative role for toxic metabolites in our system. (These findings were consistent with our previous report showing that excess tryptophan overcomes suppression by IDO-expressing macrophages ( 6) and also with reports by others using a variety of cell types ( 13, 38, 39).)
|
Finally, we asked whether IDO-mediated inhibition could occur when the concentration of tryptophan metabolites was negligible. MLRs were performed in chemically defined, serum-free medium ( 6) containing reduced levels of free tryptophan (10, 1 or 0 µM, as compared with the normal concentration in culture medium of 25–50 µM). As downstream metabolites are derived from tryptophan with a 1:1 stoichiometry, this imposed a de facto upper limit on the maximum concentration of metabolites that could be generated in the system. Fig. 8C shows that IDO + DCs continued to show full IDO-mediated inhibition even at 1 µM tryptophan, demonstrating that suppression could occur even at low levels of metabolites. In these experiments, as the concentration of tryptophan in the medium was reduced, the ability of the T cells to proliferate when they were not suppressed (i.e., in groups receiving 1MT) progressively fell, consistent with our previous reports that T cells require tryptophan for proliferation ( 6, 41).
Taken together, the experiments in Fig. 8 indicate that the production of downstream metabolites was neither necessary nor sufficient for IDO-mediated inhibition in our system, but that the tryptophan-depleting effect of IDO was required.
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
We also provide mechanistic evidence that an IDO-expressing APC can serve as a regulatory bridge connecting two independent T cell populations. In our system the CD4+ subset accounted for all the observed ability of activated T cells to induce IDO in DCs (Fig. 7). Pure CD8+ T cells (with no CD4+ cells present) did not induce IDO during MLRs and showed no evidence of IDO-mediated suppression or enhancement by 1MT. Thus, all the B7-dependent, IDO-inducing effects of activated T cells shown in Figs. 4 and 5 appeared solely mediated by the CD4+ subset. However, although the CD8+ T cells could not trigger IDO, both CD8+ and CD4+ T cells could be suppressed by IDO once it was triggered by CD4+ cells. Thus, even though CD8+ cells proliferated strongly in response to IDO+ DCs when tested in isolation, the same CD8+ cells were suppressed in the presence of CD4+ T cells (Fig. 7c). We propose that IDO thus serves as an intermediate effector mechanism in this system, allowing the CD4+ T cells to dominantly regulate the responses of CD8+ T cells via the intermediary suppressive DCs (Fig. 9). Conceptually, this is analogous to the model in which CD4+ Th cells can condition or "license" a DC so that it becomes competent to activate CD8+ T cells ( 42, 43). In our system, however, the license is to suppress rather than to activate.
|
In our system it appeared that either of the B7 counter-receptors, CTLA4 and CD28, were able to deliver a sufficient signal to trigger IDO (based on the fact that both receptors had to be blocked before inhibition was lost). However, bulk MLRs are not an ideal system in which to discriminate the biologically relevant differences between CTLA4 and CD28, because of the potential for cross-talk and bystander effects. Recombinant CD28-Ig fusion protein was significantly less efficient than CTLA4-Ig at triggering IDO (cf., Fig. 2c), which is consistent with the higher affinity of CTLA4 for B7 molecules ( 50). Functionally, CTLA4 is also known to play an important immunosuppressive and tolerogenic role in the immune system ( 51, 52), and it has been reported to be obligatory for induction of IDO in DCs by murine Tregs ( 19). Therefore, we hypothesize that CTLA4, rather than CD28, is likely to be the biologically relevant trigger for IDO in vivo.
We do not yet know which subset within the CD4+ T cell population is responsible for triggering IDO in human DCs. It is tempting to speculate that it might be the CD25+CD4+ Tregs, as has been recently suggested in mice ( 19), particularly as certain Tregs constitutively express high levels of CTLA4 ( 53, 54). However, Treg subsets in humans are less clearly defined than those in mice, and even the CD25-negative CD4+ T cells have been shown to acquire regulatory attributes after activation in vitro ( 55). In our hands, preliminary fractionation experiments have not revealed a straightforward segregation of IDO-inducing activity with the CD25+CD4+ subset in humans (unpublished observations), although improved fractionation may well reveal a specific subset of IDO-inducing cells within the larger CD4+ population. Even if there prove to be species-related differences in Treg subsets, the key point is that a population of CD4+ T cells is able to regulate other T cells, including all of the CD8+ cells, via induction of IDO in APCs.
The specific type of APC serving as the bridge in this system is likely to be a critically important biologic variable. In our in vitro culture system, we chose conditions that biased the monocyte-derived DCs toward high expression of IDO, so that suppression would predominate. Under different conditions, however, different DCs might be generated that would not express immunosuppressive IDO regardless of B7 ligation by T cells. Thus, the properties of the responding APC (i.e., whether B7 ligation leads to IDO activation) is a critical variable. In an in vitro system such as ours, the distinction between types of DCs is rather artificial, as these attributes are dictated by the culture conditions, and cultured DCs do not recapitulate the complex subsets of human DCs that exist in vivo ( 56). Nevertheless, we hypothesize that the ability to induce high levels of IDO, or not, in response to B7 ligation constitutes a real and biologically important distinction between different types of DCs in vivo. We have recently shown that there exist marked differences between different subsets of mouse DCs in their ability to up-regulate IDO when challenged by CTLA4-Ig in vivo ( 20). Whether this represents specific, committed, IDO-competent subsets of DCs, a stage of maturation at which IDO is expressed, or both remains to be determined. However, several reports suggest that IDO-expressing APCs possess potent immunosuppressive and tolerogenic properties in murine models ( 18, 19, 20, 57). Our in vitro system allows us to model at least one human counterpart of these B7-inducible, IDO-expressing DCs.
Our system for deriving such cells differs from other DC culture systems in that it has been optimized specifically to study suppression. One relevant modification includes the use of X-vivo15 medium without serum supplementation, which we and others ( 58) have observed favors adherence of a significant proportion of cells. This results in an enrichment for IDO-expressing cells in the nonadherent fraction (e.g., compared with monocytes cultured in bovine serum-based medium, where we find that the proportion of IDO+ cells is small, and almost all cells are nonadherent regardless of IDO expression ( 7)). Although there is still a 100-fold range of IDO expression by FACS in our IDO-positive cells, the relative enrichment for IDO allows suppression to predominate over activation in MLRs. This is important, because we have previously shown that when MLRs are stimulated by a mixed population of IDO+ and IDOneg DCs, it can be impossible to detect the IDO-mediated suppression against the background of predominating T cell activation unless the IDO+ cells are suitably enriched ( 7).
Even when an enriched population of IDO+ cells is available, the ability to measure T cell suppression also requires an assay system sensitive to the property of interest, as demonstrated by the importance of the sustained cell-cell contact obtained in V-bottom wells (cf., Fig. 1b). We see an identical impact of well geometry in MLR assays using mouse cells, where we know that the DCs are potently immunosuppressive in vivo ( 20). Thus, the optimized MLR assay allows us to measure a real and biologically important property; without a suitable assay, this property will be missed. This is potentially important, because increasing evidence suggests that immunoregulatory APCs can be biologically significant in vivo even when they comprise only a minority of cells. Adoptive transfer experiments in mice suggest that even a limited number of tolerogenic DCs can promote systemic tolerance ( 59). In mixing experiments, a small population of tolerogenic DCs has been reported to create systemic unresponsiveness despite coadministration of a majority of nonsuppressive DCs ( 57). And we have recently reported that activation of IDO in even a restricted subset of DCs is sufficient to systemically suppress Ag-driven T cell responses in vivo ( 20). These observations underscore the importance of having a system in which to study IDO-mediated immunoregulation in humans.
Taken together, our data suggest that IDO-mediated suppression is tightly regulated. It is contingent on the correct subset of T cells (presumably a regulatory population of CD4+ cells) sending the correct B7-mediated triggering signal to the correct (IDO-competent) subset of DCs. Only then does IDO-mediated suppression come into play. Given the potent biologic effects of a dominant immunosuppressive mechanism such as IDO, this multilayered regulation may be both appropriate and necessary.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 Address correspondence and reprint requests to Dr. David H. Munn, Medical College of Georgia, Institute of Molecular Medicine and Genetics, Augusta, GA 30912. E-mail address: dmunn{at}mail.mcg.edu
3 Abbreviations used in this paper: IDO, indoleamine 2,3-dioxygenase; CD28-Ig, CD28-Ig Fc fusion protein; CTLA4-Ig, CTLA4-Ig Fc fusion protein; DC, dendritic cell; 1MT, 1-methyl-D-tryptophan; Treg, regulatory T cell.
Received for publication July 24, 2003. Accepted for publication January 27, 2004.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
, indoleamine 2,3-dioxygenase, and tryptophan catabolism. FASEB J. 5:2516.[Abstract]
1-antitrypsin fusion antibody. Blood 102:564.-(3-benzofuranyl)-DL-alanine (the oxygen analog of tryptophan), and -[3-benzo(b)thienyl]-DL-alanine (the sulfur analog of tryptophan) are competitive inhibitors for indoleamine 2,3-dioxygenase. Arch. Biochem. Biophys. 291:326.[Medline]
RI induces the tryptophan degradation pathway involved in regulating T cell responses. J. Immunol. 169:1810.-induced indoleamine 2,3 dioxygenase and inducible nitric oxide syntahse in the replication of human cytomegalovirus in retinal pigment epithelial cells. J. Immunol. 162:957.+ dendritic cells expressing indoleamine 2,3-dioxygenase. J. Immunol. 167:708.This article has been cited by other articles:
|
|
 |
|
  F. Qian, J. Villella, P. K. Wallace, P. Mhawech-Fauceglia, J. D. Tario Jr., C. Andrews, J. Matsuzaki, D. Valmori, M. Ayyoub, P. J. Frederick, et al. Efficacy of Levo-1-Methyl Tryptophan and Dextro-1-Methyl Tryptophan in Reversing Indoleamine-2,3-Dioxygenase-Mediated Arrest of T-Cell Proliferation in Human Epithelial Ovarian Cancer Cancer Res., July 1, 2009; 69(13): 5498 - 5504. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Huang, P. Johnston, B. Zhang, A. Zakari, T. Chowdhry, R. R. Smith, E. Marban, H. Rabb, and K. L. Womer Kidney-Derived Stromal Cells Modulate Dendritic and T Cell Responses J. Am. Soc. Nephrol., April 1, 2009; 20(4): 831 - 841. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 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]
|
|
|
|
 |
|
 A. J. Muller, M. D. Sharma, P. R. Chandler, J. B. DuHadaway, M. E. Everhart, B. A. Johnson III, D. J. Kahler, J. Pihkala, A. P. Soler, D. H. Munn, et al. Chronic inflammation that facilitates tumor progression creates local immune suppression by inducing indoleamine 2,3 dioxygenase PNAS, November 4, 2008; 105(44): 17073 - 17078. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 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]
|
|
|
|
 |
|
 K. Botturi, Y. Lacoeuille, P. Thomas, S. Boniface, M. Reynaud-Gaubert, and A. Magnan CTLA-4-mediated regulatory phenotype of T-cells in tolerant lung recipients Eur. Respir. J., June 1, 2008; 31(6): 1167 - 1176. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Kang, S. J. Huddleston, J. M. Fraser, and A. Khoruts De novo induction of antigen-specific CD4+CD25+Foxp3+ regulatory T cells in vivo following systemic antigen administration accompanied by blockade of mTOR J. Leukoc. Biol., May 1, 2008; 83(5): 1230 - 1239. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Lehner, A. Stilper, P. Morhart, and W. Holter Plasticity of dendritic cell function in response to prostaglandin E2 (PGE2) and interferon-{gamma} (IFN-{gamma}) J. Leukoc. Biol., April 1, 2008; 83(4): 883 - 893. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
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]
|
|
|
|
|
|
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]
|
|
|
|
 |
|
 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]
|
|
|
|
 |
|
 A. Boasso, M. Vaccari, A. Hryniewicz, D. Fuchs, J. Nacsa, V. Cecchinato, J. Andersson, G. Franchini, G. M. Shearer, and C. Chougnet Regulatory T-Cell Markers, Indoleamine 2,3-Dioxygenase, and Virus Levels in Spleen and Gut during Progressive Simian Immunodeficiency Virus Infection J. Virol., November 1, 2007; 81(21): 11593 - 11603. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
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]
|
|
|
|
|
|
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]
|
|
|
|
|
|
P. Feunou, S. Vanwetswinkel, F. Gaudray, M. Goldman, P. Matthys, and M. Y. Braun Foxp3+CD25+ T Regulatory Cells Stimulate IFN-{gamma}-Independent CD152-Mediated Activation of Tryptophan Catabolism That Provides Dendritic Cells with Immune Regulatory Activity in Mice Unresponsive to Staphylococcal Enterotoxin B J. Immunol., July 15, 2007; 179(2): 910 - 917. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. F. May Jr., X. Chang, H. Zhang, K. D. Lute, P. Zhou, E. Kocak, P. Zheng, and Y. Liu B7-Deficient Autoreactive T Cells Are Highly Susceptible to Suppression by CD4+CD25+ Regulatory T Cells J. Immunol., February 1, 2007; 178(3): 1542 - 1552. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D.-Y. Hou, A. J. Muller, M. D. Sharma, J. DuHadaway, T. Banerjee, M. Johnson, A. L. Mellor, G. C. Prendergast, and D. H. Munn Inhibition of Indoleamine 2,3-Dioxygenase in Dendritic Cells by Stereoisomers of 1-Methyl-Tryptophan Correlates with Antitumor Responses Cancer Res., January 15, 2007; 67(2): 792 - 801. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. A. Sarkar, R. Wong, S. I. Hackl, O. Moua, R. G. Gill, A. Wiseman, H. W. Davidson, and J. C. Hutton Induction of Indoleamine 2,3-Dioxygenase by Interferon-{gamma} in Human Islets Diabetes, January 1, 2007; 56(1): 72 - 79. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. L. Seymour, V. Ganapathy, A. L. Mellor, and D. H. Munn A high-affinity, tryptophan-selective amino acid transport system in human macrophages J. Leukoc. Biol., December 1, 2006; 80(6): 1320 - 1327. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. Zhu, F. Ji, Y. Wang, Y. Zhang, Q. Liu, J. Z. Zhang, K. Matsushima, Q. Cao, and Y. Zhang Synovial Autoreactive T Cells in Rheumatoid Arthritis Resist IDO-Mediated Inhibition J. Immunol., December 1, 2006; 177(11): 8226 - 8233. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Bharat, R. C. Fields, E. P. Trulock, G. A. Patterson, and T. Mohanakumar Induction of IL-10 Suppressors in Lung Transplant Patients by CD4+25+ Regulatory T Cells through CTLA-4 Signaling J. Immunol., October 15, 2006; 177(8): 5631 - 5638. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. S. von Bergwelt-Baildon, A. Popov, T. Saric, J. Chemnitz, S. Classen, M. S. Stoffel, F. Fiore, U. Roth, M. Beyer, S. Debey, et al. CD25 and indoleamine 2,3-dioxygenase are up-regulated by prostaglandin E2 and expressed by tumor-associated dendritic cells in vivo: additional mechanisms of T-cell inhibition Blood, July 1, 2006; 108(1): 228 - 237. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Stasiolek, A. Bayas, N. Kruse, A. Wieczarkowiecz, K. V. Toyka, R. Gold, and K. Selmaj Impaired maturation and altered regulatory function of plasmacytoid dendritic cells in multiple sclerosis Brain, May 1, 2006; 129(5): 1293 - 1305. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. Degauque, D. Lair, A. Dupont, A. Moreau, G. Roussey, F. Moizant, F. X. Hubert, C. Louvet, M. Hill, F. Haspot, et al. Dominant Tolerance to Kidney Allografts Induced by Anti-Donor MHC Class II Antibodies: Cooperation between T and Non-T CD103+ Cells J. Immunol., April 1, 2006; 176(7): 3915 - 3922. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Orabona, P. Puccetti, C. Vacca, S. Bicciato, A. Luchini, F. Fallarino, R. Bianchi, E. Velardi, K. Perruccio, A. Velardi, et al. Toward the identification of a tolerogenic signature in IDO-competent dendritic cells Blood, April 1, 2006; 107(7): 2846 - 2854. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. Gottfried, L. A. Kunz-Schughart, S. Ebner, W. Mueller-Klieser, S. Hoves, R. Andreesen, A. Mackensen, and M. Kreutz Tumor-derived lactic acid modulates dendritic cell activation and antigen expression Blood, March 1, 2006; 107(5): 2013 - 2021. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Harnaha, J. Machen, M. Wright, R. Lakomy, A. Styche, M. Trucco, S. Makaroun, and N. Giannoukakis Interleukin-7 Is a Survival Factor for CD4+ CD25+ T-Cells and Is Expressed by Diabetes-Suppressive Dendritic Cells Diabetes, January 1, 2006; 55(1): 158 - 170. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S.-Y. Tseng, M. Liu, and M. L. Dustin CD80 Cytoplasmic Domain Controls Localization of CD28, CTLA-4, and Protein Kinase C{theta} in the Immunological Synapse J. Immunol., December 15, 2005; 175(12): 7829 - 7836. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. Miwa, S. Hayakawa, S. Miyazaki, S. Myojo, Y. Sasaki, M. Sakai, O. Takikawa, and S. Saito IDO expression on decidual and peripheral blood dendritic cells and monocytes/macrophages after treatment with CTLA-4 or interferon-{gamma} increase in normal pregnancy but decrease in spontaneous abortion Mol. Hum. Reprod., December 1, 2005; 11(12): 865 - 870. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F. Marteau, N. S. Gonzalez, D. Communi, M. Goldman, J.-M. Boeynaems, and D. Communi Thrombospondin-1 and indoleamine 2,3-dioxygenase are major targets of extracellular ATP in human dendritic cells Blood, December 1, 2005; 106(12): 3860 - 3866. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. J. M. van Oosterhout and N. Bloksma Regulatory T-lymphocytes in asthma Eur. Respir. J., November 1, 2005; 26(5): 918 - 932. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. V. Parry, J. M. Chemnitz, K. A. Frauwirth, A. R. Lanfranco, I. Braunstein, S. V. Kobayashi, P. S. Linsley, C. B. Thompson, and J. L. Riley CTLA-4 and PD-1 Receptors Inhibit T-Cell Activation by Distinct Mechanisms Mol. Cell. Biol., November 1, 2005; 25(21): 9543 - 9553. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Bhatia, M. Edidin, S. C. Almo, and S. G. Nathenson Different cell surface oligomeric states of B7-1 and B7-2: Implications for signaling PNAS, October 25, 2005; 102(43): 15569 - 15574. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 O. Pino, M. Martin, and S. M. Michalek Cellular Mechanisms of the Adjuvant Activity of the Flagellin Component FljB of Salmonella enterica Serovar Typhimurium To Potentiate Mucosal and Systemic Responses Infect. Immun., October 1, 2005; 73(10): 6763 - 6770. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. Braun, R. S. Longman, and M. L. Albert A two-step induction of indoleamine 2,3 dioxygenase (IDO) activity during dendritic-cell maturation Blood, October 1, 2005; 106(7): 2375 - 2381. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. Potula, L. Poluektova, B. Knipe, J. Chrastil, D. Heilman, H. Dou, O. Takikawa, D. H. Munn, H. E. Gendelman, and Y. Persidsky Inhibition of indoleamine 2,3-dioxygenase (IDO) enhances elimination of virus-infected macrophages in an animal model of HIV-1 encephalitis Blood, October 1, 2005; 106(7): 2382 - 2390. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Z. Fehervari and S. Sakaguchi CD4+ regulatory cells as a potential immunotherapy Phil Trans R Soc B, September 29, 2005; 360(1461): 1647 - 1661. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Dercamp, K. Chemin, C. Caux, G. Trinchieri, and A. P. Vicari Distinct and Overlapping Roles of Interleukin-10 and CD25+ Regulatory T Cells in the Inhibition of Antitumor CD8 T-Cell Responses Cancer Res., September 15, 2005; 65(18): 8479 - 8486. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. A Wallet, P. Sen, and R. Tisch Immunoregulation of Dendritic Cells Clin. Med. Res., August 1, 2005; 3(3): 166 - 175. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
U. Hainz, P. Obexer, C. Winkler, P. Sedlmayr, O. Takikawa, H. Greinix, A. Lawitschka, U. Putschger, D. Fuchs, S. Ladisch, et al. Monocyte-mediated T-cell suppression and augmented monocyte tryptophan catabolism after human hematopoietic stem-cell transplantation Blood, May 15, 2005; 105(10): 4127 - 4134. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Loser, A. Scherer, M. B. W. Krummen, G. Varga, T. Higuchi, T. Schwarz, A. H. Sharpe, S. Grabbe, J. A. Bluestone, and S. Beissert An Important Role of CD80/CD86-CTLA-4 Signaling during Photocarcinogenesis in Mice J. Immunol., May 1, 2005; 174(9): 5298 - 5305. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Andersson, A. Boasso, J. Nilsson, R. Zhang, N. J. Shire, S. Lindback, G. M. Shearer, and C. A. Chougnet Cutting Edge: The Prevalence of Regulatory T Cells in Lymphoid Tissue Is Correlated with Viral Load in HIV-Infected Patients J. Immunol., March 15, 2005; 174(6): 3143 - 3147. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. H. Munn, A. L. Mellor, M. Rossi, and J. W. Young Dendritic cells have the option to express IDO-mediated suppression or not Blood, March 15, 2005; 105(6): 2618 - 2618. [Full Text] [PDF]
|
|
|
|
|
|
S. Bozza, F. Fallarino, L. Pitzurra, T. Zelante, C. Montagnoli, S. Bellocchio, P. Mosci, C. Vacca, P. Puccetti, and L. Romani A Crucial Role for Tryptophan Catabolism at the Host/Candida albicans Interface J. Immunol., March 1, 2005; 174(5): 2910 - 2918. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Boasso, J.-P. Herbeuval, A. W. Hardy, C. Winkler, and G. M. Shearer Regulation of indoleamine 2,3-dioxygenase and tryptophanyl-tRNA-synthetase by CTLA-4-Fc in human CD4+ T cells Blood, February 15, 2005; 105(4): 1574 - 1581. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. L. Mellor, P. Chandler, B. Baban, A. M. Hansen, B. Marshall, J. Pihkala, H. Waldmann, S. Cobbold, E. Adams, and D. H. Munn Specific subsets of murine dendritic cells acquire potent T cell regulatory functions following CTLA4-mediated induction of indoleamine 2,3 dioxygenase Int. Immunol., October 1, 2004; 16(10): 1391 - 1401. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
), 200 µM 1MT, or 250 µM L-tryptophan (10x TRP).
), or F(ab')2 goat anti-mouse IgG (G
). Groups 1 and 2 are controls showing IDO-mediated inhibition of T cell proliferation and its reversal by 1MT. Group 3 shows abrogation of inhibition by blockade of CTLA4 and CD28. Group 4 shows the effect of anti-CD80 and -CD86 mAbs; a modest IDO-inducing effect was seen in the absence of exogenous cross-linking (dotted arrow), but cross-linking was required to induce full inhibition of T cell proliferation (solid arrow).
