The current study demonstrates for the first time a mechanistic link between IDO, functional activation of Tregs, and the PD-1/PD-L pathway. Each of these mechanisms is known to be independently important in tumor immunology, and strategies targeting each mechanism are currently in clinical trials or in active preclinical development. We now show that these 3 powerful regulatory mechanisms are tightly linked at the level of Treg activation in the TDLN. This linked pathway constitutes a major contributor to the intensely immunosuppressive milieu present in TDLNs. Since this suppressive milieu drives T cell anergy and unresponsiveness to tumor antigens presented in the TDLNs (32
), identification of molecular mechanisms contributing to this suppression represents an important goal in cancer immunotherapy.
Our findings suggest a hypothetical model (Figure ) in which IDO-induced Treg activation proceeds via a self-amplifying loop. We hypothesize that when IDO+
pDCs present antigen to effector T cells in the presence of mature, resting Tregs, this initiates a GCN2-dependent activation of the Tregs by IDO. In other cells, GCN2 is known to activate a downstream stress-response pathway, resulting in a coordinated program of changes in gene expression (26
). In the case of CD8+
effector T cells, we have shown that activation of the GCN2 pathway leads to cell cycle arrest and anergy (9
). In the case of Tregs, we now show that GCN2 signaling is critical for allowing IDO-induced functional activation. Based on our data (Figure A and Supplemental Figure 5), we speculate that the activating Tregs reciprocally induce high levels of IDO in pDCs, via CTLA4-B7 interaction (27
), leading to increased production of tryptophan metabolites. These metabolites then complete the full activation of the Tregs (as suggested by data shown in Figure C and Supplemental Figure 6), resulting in emergence of the novel, highly potent PD-1/PD-L–dependent form of suppression that we describe. This model is speculative, but it is consistent with our data, and the 2 key features of the model are clear and well supported: direct IDO-induced activation of mature Tregs and PD-1/PD-L–dependent suppression by IDO-activated Tregs.
Proposed hypothetical model of IDO-induced Treg activation based on synthesis of results from the in vitro models.
A role for PD-1/PD-L as a downstream suppressor mechanism for Tregs has not been previously described. However, induction of a another suppressive B7 family member, B7-H4, on APCs by Tregs has been recently shown (25
). In our system, the PD-1/PD-L mechanism of suppression was only found with the IDO-induced form of Treg activation and was not seen with the widely studied anti-CD3-induced form of Treg activation. The PD-1/PD-L pathway has been the focus of considerable interest because it has been found to mediate clonal exhaustion and T cell anergy in HIV and other chronic viral infections (34
) as well as tolerance to self antigens and immune suppression in cancer (35
). Our findings provide a novel mechanistic link between the PD-1/PD-L system, Tregs, and IDO.
Tregs isolated from TDLNs in vivo were constitutively activated, displaying spontaneous suppressor activity that was as potent as the highest levels reported for Tregs extensively activated in vitro (15
). The majority of this constitutive Treg activity in TDLNs was mediated via the novel IDO-induced, PD-1/PD-L–dependent mechanism. We demonstrate the existence of 2 distinct, clearly distinguishable forms of Treg activity — the conventional form elicited by anti-CD3 crosslinking, in which suppression was dependent on IL-10/TGF-β, was reversed by IL-2, and was unaffected by PD-1/PD-L blockade; and the novel IDO-induced form, which was not dependent on IL-10/TGF-β, was not reversed by IL-2, and was strictly dependent on the PD-1/PD-L pathway. Under IDO-sufficient conditions, 75%–90% of the constitutive Treg activity in TDLNs was due to the IDO-induced form of Treg activity. This IDO-induced component was completely lost when tumors were grown in IDO-KO mice or in mice treated with an IDO-inhibitor drug during tumor growth. Under these chronically IDO-deficient conditions, tumors showed a compensatory increase in the form of Treg activity that was not dependent on IDO, consistent with emergence of tumor escape variants (36
). However, while tumors were thus able to compensate for artificial genetic or pharmacologic ablation of IDO, from a clinical standpoint human patients would normally be IDO sufficient. Thus, the key observation in our system was that 75%–90% of the naturally occurring Treg activity in TDLNs was of the IDO-induced, PD-1/PD-L–dependent form.
In vitro, IDO activity also promoted de novo upregulation of Foxp3 expression in naive CD4+
T cells. This finding is not novel, since the pathway has already been described by Fallarino and colleagues (11
). In our system, the mature, preexisting Tregs activated by IDO were 100-fold more potent on a per-cell basis than the newly differentiated Foxp3+
cells. In human T cells, it is known that Foxp3 upregulation does not necessarily connote stable commitment to Treg differentiation (37
), so it is possible that not all of the newly derived Foxp3+
cells would go on to become Tregs. Nevertheless, it is relevant to note that IDO is potentially linked to the Treg lineage at 2 points, the rapid and potent activation of mature Tregs that we describe and the potential for de novo differentiation of new Tregs.
IDO-induced Treg activation was almost entirely prevented by blockade of CTLA4. CTLA4 has multiple regulatory roles in the immune system, most of which are intrinsic to the CTLA4+
T cells themselves; however, it is also known that CTLA4 can induce IDO expression in DCs, via back-signaling through B7 molecules (27
). We hypothesize that CTLA4 on Tregs delivers a signal to IDO+
pDCs that enhances their normal level of IDO enzymatic activity and thus increases the production of immunoregulatory metabolites. Interpretation of such studies is complex because it is difficult to separate cell-autonomous effects of the antibody on Treg function from the effect of the antibody on IDO, so further studies are required. However, from a therapeutic standpoint, anti-CTLA4 antibodies are in late-stage clinical trials (39
), so it is of interest to note that CTLA4 blockade also interrupts the novel IDO/Treg/PD-L pathway.
The human counterpart of the IDO+
pDCs in mouse TDLNs is not yet established, and human and mouse DC subsets do not always correspond. However, a prominent population of IDO-expressing cells is observed in many human TDLNs (40
), displaying a characteristic plasmacytoid morphology (41
). Recently, human plasmacytoid DCs (CD123+
) have been shown to upregulate IDO in response to HIV infection (42
); thus, authentic human pDCs can be induced to express IDO. Further investigation will be required to specifically identify the IDO-expressing cells found in human TDLNs. Future studies will also be needed to address the possible developmental roles of IDO and GCN2 in the differentiation of the Treg lineage. Preliminary studies demonstrate selective but significant functional defects in Tregs derived from IDO-KO, GCN2-KO, and CHOP-KO mice, suggesting that the IDO pathway may have broader importance for aspects of normal Treg differentiation.
The current study suggests that patients with cancer may have abnormally increased Treg activity in TDLNs, due in part to the effects of IDO. Once tumors are established, simply blocking IDO was not sufficient to fully reverse the suppressive milieu in the TDLN (Figure C). But even in established tumors, blocking IDO allowed initial activation of tumor-specific effector T cells in TDLNs, with attempted cell division. Combining IDO-inhibitor drugs with chemotherapy may further help to reverse the established suppressive milieu in TDLNs. Therapeutic strategies to block IDO, tumor-induced Tregs, and the PD-1/PD-L pathway are all currently in clinical or preclinical development. Our demonstration of a molecular link uniting all 3 of these potent immunosuppressive mechanisms may have significant implications for cancer immunotherapy.