The data exposed in this article suggest the existence of a linear pathway that links tumor progression to immune suppression. Tumor cells produce factors that stimulate local IMDCs to produce TGF-β. TGF-β then acts as a co-stimulator to drive the proliferation of FOXP3+CD4+CD25+ T reg cells in a TGF-βRII–dependent fashion, and T reg cells in turn mediate immunosuppression. This pathway has been unraveled in two distinct rodent species, enhancing the likelihood (but by no means proving) that it also applies to the human system.
During tumor progression, T reg cells accumulate in tumors and secondary lymphoid organs of humans (13
). It has been suggested that chemokines produced by tumor cells or tumor-infiltrating macrophages recruit T reg cells into the tumor bed (17
). On theoretical grounds, T reg cell accumulation could also result from the priming and differentiation of naive CD4+
T lymphocytes into T reg cells, a phenomenon well established in vitro (22
) and on homeostatic proliferation in vivo (25
). Here, we formally demonstrated that the T reg cell accumulation in tumor-draining lymphoid organs largely resulted from their local proliferation (as opposed to reduced apoptosis or nonproliferative differentiation). These results are in line with the seminal work of North and Awwad, who highlighted the expansion of cycling CD4+
suppressor cells in tumors and their role in tumor-induced immune tolerance (26
T reg cell proliferation may be related to IL-2 (11
) and MHC class II peptide complexes (28
), as well as TGF-β (20
). We retain that it is unlikely that IL-2 drives T reg cell proliferation stimulated by IMDCs during tumor growth. Indeed, to date, DCs have solely been reported to produce IL-2 after LPS stimulation, bacterial infection, or CD40 ligation (27
), conditions that are unlikely to be met in tumor-bearing hosts. Reportedly, immunization with MHC class II epitopes in TCR transgenic mice promoted the expansion of peptide-specific T reg cells in vivo (28
). Similarly, MHC class II peptide–pulsed mature DCs could elicit peptide-specific T reg cells capable of preventing the onset of diabetes (9
). Our data suggest that IMDC-mediated T reg cell expansion occurring in tumor-bearing hosts is MHC class II restricted because T reg cell proliferation induced by IMDCs from TBR was abolished by the depletion of MHC class II+
cells ( C). Moreover, T reg cell proliferation could be elicited by a combination of anti-CD3 mAb (which mimics the MHC class II peptide complex) and TGF-β co-stimulation ( C). Peng et al. (20
) first unraveled the role of TFG-β in inducing T reg cell proliferation for the control of diabetes. Working in a model of dextran sulfate–induced autoimmune colitis, Huber et al. (21
) confirmed the requirement for TGF-βRII signaling in T reg cells for their proliferation in vivo. However, the source of TGF-β was either transfected pancreatic islet β cells (20
) or not identified (21
). Here, we demonstrate that a specific subset of DC-infiltrated lymphoid organs during tumor progression provides a source of TGF-β ().
The role of TGF-β in the regulation of T reg cell proliferation in tumor-bearing hosts has not been reported so far. An immunosuppressive effect and a tumor-enhancing role of TGF-β has been demonstrated in the model of syngeneic mice bearing T cells defective in TGF-βRII signaling (30
), but it was assumed that TGF-β produced by tumor cells exerted a direct suppressive effect on the antitumor-specific T cells (30
). We provide compelling evidence indicating that TGF-β produced by tumor cell–licensed IMDCs was critical for the proliferation and accumulation of T reg cells. Indeed, IMDCs isolated from tumor bearers, but not from TFR or TFM, elicited T reg cell proliferation () in a TGF-β–dependent fashion (). Moreover, IMDCs from TFM were converted to a T reg cell-stimulatory phenotype when licensed by tumor cells ( and ).
Our experiments using positive () or negative selection ( C) of either CD11c+/CD11b+ cells or MHC class II+ cells suggest that IMDCs are essential for the proliferation of T reg cells in vitro. This particular DC population was CD11c+/I-Ab+ in mice and OX42+/OX17+ in rats, coexpressed CD11b, and exhibited low levels of co-stimulatory molecules. Several arguments support the relevance of these IMDC for the expansion of T reg cells in tumor-bearing animals. First, both IMDCs and T reg cells resided and expanded in tumor DLNs during tumor progression ( and ). IMDCs expanded by fivefold and T reg cells by twofold by day 15 or 28 in DLNs of animals with B16F10 and PROb tumors, respectively. Second, IMDCs isolated from tumor bearers induced T reg cell proliferation when adoptively transferred into tumor-free hosts (). Third, the IMDC-mediated T reg cell priming depended on both MHC class II and TGF-β molecules ().
Mature DCs expressing high levels of MHC class II and CD80 (9
), as well as immature DCs (31
), have been reported to be mitogenic for T reg cells. In the two rodent tumor cells that we investigated in this study, we failed to detect significant (P > 0.10) numbers of completely mature DCs in tumor DLNs (unpublished data), suggesting that IMDCs are indeed the preponderant T reg cell stimulators. In line with this consideration, immature DCs were shown to produce TGF-β more actively than mature DCs (32
). Moreover, the phenotype of IMDCs is clearly different from that of indoleamine 2,3-dioxygenase–expressing plasmacytoid DCs that also may exert immunosuppressive functions (33
Tumor cells licensed DCs to promote T reg cell proliferation via TGF-β ( and ). This licensing effect involved soluble factors produced by B16F10 and PROb tumor cells. In this context, it appears intriguing that tumors reportedly produce a variety of distinct cytokines, including vascular endothelial growth factor, macrophage CSF, granulocyte-macrophage CSF, IL-6 and IL-10, capable of blocking DC differentiation, for instance through activation of STAT-3 and inactivation of the NF-κB signaling pathway (34
). Whether such soluble factors could also induce TGF-β expression and secretion by DCs remains to be assessed.
Irrespective of these questions, the data outlined in this paper delineate a novel pathway linking tumor expansion to immunosuppression. Several elements among this pathway are new (such as IMDCs stimulating T reg cell proliferation) or are newly placed (such as TGF-β as a co-stimulatory factor for T reg cells). Future research will address the question as to whether interruption of this cascade may have positive consequences for the clinical management of human cancer.