In their study, Moffett et al. attempted to detect IDO activity at the single-cell level based on immunohistological staining of quinolinic acid, which is the terminal metabolite of the enzymatic pathway initiated by IDO (24
). Their findings indicated that IDO-producing cells are distributed in lymphoid tissues and sporadically in other tissues, and have a macrophage- or DC-like appearance (3
). Recent evidence for expression of IDO in DCs came from the work of Hwu et al. who induced IDO production in human DCs by stimulation with IFN-γ and CD40 ligand (25
). Although it is generally known that DCs are potent activators of T cells, they may also play a pivotal role in the induction of peripheral tolerance (26
). Because IDO is known to reduce T cell proliferation, it is tempting to speculate that IDO-expressing DCs have suppressive properties. In their studies, Hwu et al. (25
) inhibited the proliferation of OKT3-stimulated autologous T cells with IDO-producing DCs. In another study, stimulation of CD8α+
DCs with IFN-γ, which is known to promote IDO production, enhanced their tolerogenic activity toward CD4 T cells (27
). These as well as similar findings obtained with macrophages (14
) fostered the hypothesis that expression of IDO in antigen-presenting cells confers the ability to suppress “unwanted” T cells, particularly autoreactive ones (14
), and thereby contributes to peripheral tolerance against self-antigens. Indirect proof for the role of IDO in suppression of alloreactive T cells was obtained by Munn et al. (15
) who showed that inhibition of IDO resulted in rejection of the fetus in pregnant mice. In the current series of experiments, we present a model for studying the effect of IDO on allogeneic T cells by transgenetically expressing the IDO gene in human DCs and coincubating these cells with allogeneic T cells. IDO-expressing DCs reduced tryptophan, increased kynurenine, and (as expected) suppressed the proliferation of allogeneic T cells.
Munn et al. (3
) found that IDO-expressing macrophages catabolize virtually the entire amount of tryptophan in a cell culture system comparable to ours. Although in the experiment presented herein IDO-expressing DCs decreased the tryptophan concentration from a mean of 25.73 μM to a mean of 15.64 μM, it should be mentioned that variations down to unmeasurable tryptophan concentrations were noted in other cell cultures, a phenomenon obviously due to different activities of the IDO gene. Such very low tryptophan concentrations can impair T cell proliferation. However, we also noted T cell suppression at measurable tryptophan concentrations, provided that IDO was expressed in DCs. According to the tryptophan titration curve, such “measurable” concentrations are not low enough to impair T cell proliferation. This latter observation argues against tryptophan deficiency being the only factor responsible for suppressed T cell proliferation in our cell cultures. For tryptophan deficiency as a mechanism responsible for IDO-induced T cell suppression in vivo, it must be postulated that concentrations <0.5–1 μM (14
) are generated and maintained for a longer time in certain microenvironments. Since plasma levels are in the range of 50–100 μM and never reach such low concentrations, and since the diffusion of tryptophan into tissues evidently is faster than the local degradation rate, this proposed mechanism appears questionable (14
Surprisingly, in contrast to previous speculations (3
), we found that kynurenine suppresses the T cell response. Inhibitory concentrations in the kynurenine titration experiments were 157–553 μM, which was strikingly higher than the 12.5 μM average concentration found in our cultures of IDO-expressing DCs. Therefore, at the first glance, kynurenine did not provide a plausible explanation for T cell suppression by IDO-expressing DCs. However, it is well known that besides kynurenine, IDO-induced degradation of tryptophan results in additional catabolites. Consequently, kynurenine does not act alone but in the presence of other metabolites. As shown by our experiments, 3-hydroxykynurenine and 3-hydroxyanthranilic acid are also T cell suppressive and have an additive effect when mixed with kynurenine. When 15 μM kynurenine, a concentration which does not result in any T cell suppression by itself, acts in concert with the same amounts of all other metabolites (or 3-hydroxykynurenine and 3-hydroxyanthranilic acid only), the combined activity suppresses the T cell response. It should also be mentioned that Grohmann et al. (29
) found considerably higher kynurenine concentrations in their IFN-γ–stimulated CD8α+
DC cultures than those noted by us.
Of course, it is difficult to extrapolate from observations in cell cultures to conditions in vivo. The question must be posed, however, whether the inhibitory kynurenine concentrations found in our study are comparable to those present in vivo. Normal serum kynurenine concentrations in humans range from 1 to 3 μM (22
). This is far below the inhibitory levels observed in our experiments, a finding which is not surprising because under normal conditions blood lymphocytes are not suppressed. Low blood levels, however, do not exclude the existence of high levels of kynurenine and its derivatives at defined sites in the body. It is known that IDO production in tissues is limited to certain cells (3
). During pregnancy, for instance, IDO is expressed in the placenta and creates a site of local immunosuppression toward fetal tissue (15
), although pregnant women are not generally immunosuppressed and their serum shows no change in kynurenine concentrations (28
). In one study, quinolinic acid, the terminal kynurenine metabolite, was measured in the brain and blood of HIV-infected patients. In brain, this metabolite was elevated >300-fold. There were no significant correlations, however, between serum quinolinic acid levels and those in cerebrospinal fluid or brain parenchyma (30
). Evidently, the serum does not necessarily reflect the concentrations at the site of production. On the other hand, under certain circumstances, kynurenine metabolites may dramatically increase even in serum or urine. Cancer patients, for instance, treated with IFN type I or type II, a cytokine which induces IDO, have decreased serum tryptophan levels and 5–500-fold increased urinary kynurenine metabolite concentrations (31
). Serum of AIDS patients showed 37-fold higher quinolinic acid levels than serum of healthy controls (30
). In experiments in sheep, a species known to have kynurenine concentrations comparable to those of humans, tryptophan loading resulted in increased plasma concentration of 118 ± 79.7 μM kynurenine in pregnant as well as in nonpregnant animals, whereas the concentration reached an even higher peak (247.9 ± 86.7 μM) in fetal plasma (32
). These levels are in the range found to be suppressive in our cell cultures and suggest that, under certain circumstances, T cell inhibitory kynurenine concentrations can be achieved in vivo. Two important points must be considered when comparing in vitro kynurenine data with conditions in vivo. First, our findings show that not only kynurenine but other metabolites too, namely 3-hydroxykynurenine and 3-hydroxyanthranilic acid, suppress the T cell response. Interestingly, in studies concerning the neurotoxic effect of tryptophan metabolites it was shown that these compounds have an additive effect (33
). Our data show that the T cell suppressive effect of several tryptophan metabolites is additive in vitro. If this phenomenon also applies to conditions in vivo, relatively low kynurenine concentrations may be sufficient for suppression of the T cell response. Second, in the same series of experiments dealing with the toxic effect of tryptophan metabolites on nerve cells, it was shown that the neurotoxic threshold of 3-hydroxykynurenine significantly decreased by prolonging the exposure time (33
). This finding fits with our observation that the tryptophan metabolite mixture significantly increases its cytotoxic effect on lymphocytes with every additional day of incubation. Under in vivo conditions, such as AIDS, pregnancy, etc., the exposure time is obviously longer than the duration of cell cultures used in the present study and lower kynurenine concentrations might therefore be effective.
Our experiments indicate that the mechanism of T cell suppression by kynurenine and some of its derivatives is cell death. This explains the lack of restimulation of kynurenine-suppressed T cells. Interestingly, it was shown (35
) that macrophages, cells belonging to the IDO-expressing subpopulation (14
), can promote apoptosis of peripheral blood T cells. Moreover, treatment of DCs with IFN-γ increases their IDO activity and confers tolerogenic properties apparently by initiating apoptosis of antigen-specific CD4 cells (27
). Our findings suggest that the latter phenomenon is induced by kynurenine and its derivatives, which means that these metabolites are possible mediators of inhibitory DCs. As shown in our experiments activated T cells are more susceptible to the cytotoxic action of kynurenine metabolites than resting cells. At high concentrations and longer exposure times, however, both activated and nonactivated T cells die. Translated to allogeneic T cell stimulation, that means that donor-specific (activated) clones die, whereas other (resting) clones are not (or less) harmed when exposed to kynurenine derivatives. Our restimulation experiments support this conclusion. The observation concerning induction of T cell death by kynurenine is in line with previous findings on the cytotoxic action of tryptophan metabolites on nerve cells. These compounds are known to be increased in the central nervous system in certain neurological diseases (33
). In a recent publication, Chiarugi et al. (33
) describe the induction of cell death in cortical neurons by 3-hydroxykynurenine and quinolinic acid, whereby the former apparently induces apoptosis and the latter necrosis. Quinolinic acid has also been considered a possible mediator of neurological dysfunction in AIDS (40
). Surprisingly, our experiments show that only 3-hydroxykynurenine but not quinolinic acid induces death of lymphocytes.
If functional in vivo, the mechanism delineated by our findings might have far reaching consequences for immunoregulation and for the pathogenesis of certain diseases. As already mentioned, IDO is involved in suppression of fetal rejection and possibly in control of autoreactive T cells (3
). According to our observations, this suppression might be mediated by tryptophan metabolites which result from increased IDO activity. Consequently, high levels of such metabolites in the fetal-maternal interface may constitute a “gateway to inferno” which sentences to death all T cells which pass through. Interestingly, our findings show that not only T cells but also B and NK cells are affected. In contrast, DCs, the cells which produce IDO, are resistant to tryptophan metabolite cytotoxicity.
IDO-mediated suppression of the immune response obtains special relevance in patients with HIV infection. Selected strains of HIV-1 are capable of inducing IDO synthesis with subsequent tryptophan metabolism in human macrophages, a process apparently triggered by transient production of IFN-γ (41
). In line with these observations are clinical studies showing that the concentration of serum kynurenine increases and that of tryptophan decreases with immune stimulation in HIV-infected patients, resulting in a high kynurenine/tryptophan ratio. Most importantly, this ratio shows a reciprocal relationship to the CD4 count and the stage of the disease (42
). In the light of our findings, it is tempting to speculate that tryptophan metabolites induce T cell death and thus contribute to the immunsosuppression observed in HIV-infected patients.
If the immune system “uses” IDO-producing DCs for suppression of “unwanted” immune reactions, the same strategy could be attempted for induction of tolerance in a transplant setting. It has already been shown that donor DC progenitors, when injected into the prospective transplant recipient, prolong graft survival, conceivably by inactivating donor-reactive T cells (43
). Our study points out a way for deliberately generating “suppressive” donor DCs which can be used for tolerance induction in transplant recipients. Conceivable alternatives would be the direct expression of the IDO-gene in the graft or the use of tryptophan metabolites as immunosuppressive agents. The latter therapeutic strategy is particularly interesting in the light of our observation that tryptophan metabolites preferentially kill activated T cells, the cells which mediate graft rejection.
In conclusion, our experiments show that DCs which transgenetically express IDO are able to lower the tryptophan concentration, increase the kynurenine concentration, and suppress the allogeneic T cell response. The tryptophan metabolites kynurenine, 3-hydroxykynurenine, and 3-hydroxyanthranilic acid inhibit T cell proliferation by a time-dependent cytotoxic action, an effect which concerns mainly activated T cells, but also B and NK cells.