In the current study we identify a population of immunoregulatory DCs in TDLNs that are capable of mediating active immunosuppression in vitro, and of creating profound local T cell anergy in vivo. Previous studies of DCs isolated from tumors or TDLNs have observed a marked defect in their ability to stimulate T cells (47
), which has usually been attributed to immaturity. However, it can be difficult to distinguish immaturity from more active forms of suppression (15
). The key advance embodied in the current study is the application of specific experimental models (IDO inhibitors, IDO-KO mice, in vivo adoptive transfer) to unambiguously demonstrate the presence of active, dominant, IDO-mediated suppression in TDLNs.
An important attribute of IDO-mediated suppression was its fundamentally local nature. First, the IDO+ cells themselves were localized, being found in TDLNs but not in other LNs from the same animal. Second, adoptive-transfer studies demonstrated that complete anergy was created in the local draining LNs, even though T cells at remote sites retained reactivity. From the perspective of the tumor, we hypothesize that local anergy may be all that is required. A number of studies have shown that tumors can be locally tolerated despite the systemic presence of competent, tumor-specific T cells (5
). Such tumors grow unchecked, demonstrating that immunosuppression does not have to be systemic in order to be effective. If all the LNs draining all the sites of tumor create local anergy, then the tumor will be de facto tolerated.
In this regard, it becomes relevant that IDO-mediated suppression was dominant over other, nonsuppressive APCs. This occurred even when antigen presentation was known to be restricted solely to IDO– APCs (compare Figure ). Quantitatively, even a few percent of IDO-expressing pDCs was sufficient to suppress all T cell responses in vitro. Consistent with this, in our adoptive-transfer experiments the majority of transferred DCs were nonsuppressive (as shown by the fractionation experiments in Figure ), yet all of the BM3 cells in the recipient LNs were rendered anergic. Anergy was antigen-specific (i.e., other host T cells remained responsive to mitogen, and to antigens not expressed on the transferred DCs). The antigen-specific nature of this anergy would not be incompatible with participation of a third-party (bystander) mode of suppression. As described in other systems (55
), induction of anergy may require both an antigen-nonspecific suppressive signal, and simultaneous signaling via the TCR (conferring antigen specificity). In our system, we postulate that anergy is induced in any T cell that encounters antigen (regardless of the APC) in a milieu that is rendered immunosuppressive by IDO. Thus, we hypothesize that a small population of IDO+ pDCs is able to redefine the entire TDLN as a tolerogenic milieu, even for tumor antigens being cross-presented by other, normally immunogenic APCs.
The downstream molecular mechanisms by which IDO is able to suppress neighboring T cells remains to be elucidated. The existence of some such mechanism has been inferred from previous reports showing that even a small minority of IDO-expressing DCs can dominantly suppress T cell responses in vivo (57
). Conceptually, the suppressive effects of IDO fall into two categories: (a) those mediated by depletion of tryptophan, which include its antimicrobial (60
) and antiviral (62
) effects, and inhibition of T cell proliferation in some models (18
); and (b) effects mediated by toxic downstream metabolites of tryptophan, which include CD4+ T cell apoptosis (65
) and inhibition of T cell proliferation in other models (66
). In the case of third-party suppression, where inhibition must occur at a distance, a role for soluble factors such as tryptophan metabolites would seem intuitively likely, but experiments using the defined four-party system described in Figure should help elucidate these mechanisms.
IDO-mediated suppressor activity in TDLNs segregated with a novel population of pDCs expressing CD19, a marker of the B cell lineage (68
). It is known that early CD19+ pro-B cells can give rise to DCs in vitro (70
), and recent analyses of both human and murine pDCs have suggested a B-lymphoid origin for a subset of pDCs (72
). Despite these observations, however, most previous studies have failed to appreciate the CD19+ subset of pDCs; and where they have been described (74
), they were excluded on the assumption that they were contaminating B cells. Previously, we have reported that pDCs constitute one of the “IDO-competent” subsets of DCs, able to upregulate IDO (by immunohistochemistry) in response to CTLA4-Ig treatment (58
). In that earlier study we, like most investigators (40
), excluded CD19+ cells from our sorted pDC preparation (which we defined as CD11c+B220+CD19–). We did not realize that — although the CD19– pDCs did indeed express IDO by immunohistochemistry, as reported — we were excluding a functionally important subset of the pDCs. (In that study, suppressor activity was measured only in vivo, not on the sorted subsets.) In the current study, the development of micro-scale MLR assays allowed us to follow the functional IDO-mediated suppressor activity (not just protein expression), and hence we were able to identify the novel CD19+ pDC subset. It is not surprising that some subsets of DCs expressed IDO protein (by immunohistochemistry or Western blot) without significant suppressor activity, since this is well described in the literature (39
). Thus, the conclusion of our earlier report, that pDCs are one of the IDO-competent DC subsets, remains unchanged; but we would now expand the definition of pDCs to include the functionally important CD19+ subset.
With the exception of their B-lineage attributes, the CD19+ pDCs expressed markers consistent with murine pDCs. Some DCs can display high autofluorescence and nonspecific binding, which may complicate analysis by FACS, so we were careful to ensure that our characterization of the CD19+ pDCs also rested on functional studies, not just on marker analysis. We show that expression of CD19 and pax5 mRNA segregated with surface CD19 staining (see supplemental material), and that the CD19+ pDCs contained essentially all of the IDO-mediated suppressor activity. Thus, by functional criteria, the CD19+ pDCs represented a distinct and important subset of DCs in TDLNs.
The CD19+ pDCs were not restricted to TDLNs. They were also found in normal LNs and spleen, but in these sites they did not constitutively express IDO and were not spontaneously suppressive. We have previously shown that administration of recombinant CTLA4-Ig in vivo upregulates IDO, preferentially affecting the B220+ and CD8α+ subsets of DCs (58
). By analogy, we hypothesize that some factor in the TDLNs “pre-activates” the CD19+ pDCs for constitutive IDO-mediated suppression. This might be a microenvironmental signal (e.g., a local cytokine); or, as recently described (59
), it might be a population of IDO-inducing Treg’s. Elucidating these upstream inducing factors will be important in understanding how tumors have evolved to exploit the IDO mechanism.
The CD19+ subset of pDCs in TDLNs expressed CD123 and CCR6, which we have previously described as segregating preferentially with IDO-expressing human monocyte-derived DCs and monocyte-derived macrophages (20
). CCR6 has been described on at least a subset of murine pDCs (41
), but (unlike the case in humans) CD123 has been reported to be negative on murine pDCs. However, CD123 expression may have been missed, since we found that there was essentially no CD123 expression on pDCs from normal (non–tumor-draining) LNs (see Figure D). Even when pDCs were isolated from TDLNs, it was only the CD19+ pDCs that expressed significant levels of CD123 (and these cells are usually excluded from analysis). Thus, in mice as in humans, there may be a preferential association of CD123 and CCR6 as markers of IDO-expressing APCs. Whether the converse will prove true, that the human IDO+ DCs in TDLNs resemble their murine CD19+ counterparts, remains to be determined. Others have shown that some human pDCs do indeed appear to arise from the B cell lineage (72
), and we observe B cell markers such as CD20 on at least a subset of IDO-expressing cells in TDLNs (see Supplemental Figure ). The key point, however, is that IDO-expressing human APCs (whatever the details of their immunophenotype) are found in human TDLNs, and they appear to correlate with poor clinical outcome in the patient population studied (Figure B).
The current study demonstrates that IDO-expressing DCs can be a potent endogenous immunosuppressive mechanism in tumor-bearing mice. We know that IDO is not the only way that tumors evade the immune system, because IDO-KO mice are still susceptible to tumors, and tumors do not spontaneously regress in mice treated with 1MT alone (31
). This is hardly surprising, however, since tumors deprived of any single protective mechanism will rapidly evolve escape variants (76
). The salient point from a clinical perspective is that all human tumors will have arisen in IDO-sufficient hosts; therefore some tumors may have evolved to rely upon this mechanism for protection. The ability to acutely deprive these tumors of the protective IDO mechanism, by administering IDO-inhibitor drugs such as 1MT, may provide a therapeutic window in which to break tolerance to tumor antigens.