Our studies have uncovered an unexpected mechanism by which the Tim-3/galectin-9 pathway controls IFN-γ producing Th1 cells. Tim-3 is expressed by terminally differentiated Th1 cells which then upregulate the expression of Tim-3 ligand, galectin-9, through the production of IFN-γ (25
). Galectin-9 directly interacts with Tim-3 to inhibit the Th1-cell response by triggering cell death (15
). We now show that the Tim-3/galectin-9 pathway could also indirectly regulate Th1 immune responses through the expansion of CD11b+
cells. Interestingly, this cell surface phenotype is consistent with that described for granulocytic or PMN-like myeloid-derived suppressor cells (MDSC) (10
). In addition, our morphological analysis of CD11b+
cells further supports that these cells are granulocytic MDSC (Supplementary Fig. 2).
MDSC are a heterogeneous population of myeloid cells generally identified as being positive for CD11b and Gr-1 that expand in large numbers in tumor-bearing mice, cancer patients and after infection, trauma or autoimmunity (reviewed in (20
)). MDSC are potent suppressors of T cell immunity and their presence is correlated with poor clinical outcome in cancer. Recently, MDSC have been subdivided into two classes: monocytic (CD11b+
and granulocytic (CD11b+
). Both of these populations are suppressive for T cells but by different mechanisms. Monocytic MDSC suppress by production of nitric oxide and granulocytic MDSC suppress by a mechanism which involves IFN-γ and possibly the production of reactive oxygen species as well as arginine metabolism (10
). Indeed, IFN-γ has long been associated with the expansion and suppressive function of MDSC as MDSC fail to accumulate in IFN-γ R−/−
mice and anti-IFN-γ antibody abrogates their suppressor function (6
). In this paper we have made a novel observation that the Tim-3/galectin-9 pathway plays a role in the expansion and/or activation of granulocytic MDSC, which is consistent with previous studies implicating IFN-γ in MDSC-mediated immune suppression and that Tim-3 is a cell surface receptor expressed on IFN-γ-secreting Th1 cells.
Increased arginine metabolism acting in concert with iNOS is also implicated in MDSC-mediated immunosuppression. Consistent with this, we found that Arginase II is highly upregulated in CD11b+
cells (Supplementary Fig. 3) and that the arginase inhibitor NOR-NOHA abrogates suppression by CD11b+
cells from gal-9 Tg mice (Supplementary Fig 5). We also found that many genes that are involved in inflammation are more highly expressed in CD11b+
cells. In particular, we observed an upregulation of many IL-1/TNF-related genes, including IL-1R type II. In this regard, IL-1β has been shown to recruit MDSC in tumor tissue (28
) and to activate MDSC both in vitro
and in vivo
). Moreover, IL-1R−/−
mice exhibit a defect in MDSC accumulation (31
). The expression profile of the CD11b+
cells in our study is consistent with these observations and with the expression profile that has been reported for splenic CD11b+
cells from tumor-bearing mice (9
Cell:cell contact has long been known to be an important component in MDSC-mediated immunosuppression (reviewed in (32
)). Indeed suppression does not occur if MDSC are separated from T cells by a semi-permeable membrane, indicating the need for cell: cell contact and interaction of membrane-bound molecule(s). However, the nature of this membrane-bound molecule(s) has remained elusive. Based on our data, we propose that Tim-3 on IFN-γ-secreting T cells interacting with galectin-9 bound on MDSC is one such cell surface receptor/ligand pathway involved in MDSC expansion/function. Our observation of the increased frequency of CD11b+
cells and increased tumor growth in Tim-3 Tg mice compared to wild type littermates is in keeping with this. Several cytokines and growth factors produced by tumors, such as GM-CSF, have been shown to be involved in the generation of MDSC. The Tim-3/galectin-9 pathway may act in concert with these factors to promote MDSC expansion and suppressor function.
While our data strongly support a role for the Tim-3/galectin-9 pathway in promoting MDSC, our data in the 4T1 tumor model clearly show that CD11b+Gr-1+ cells can expand, although to a lesser degree, in Tim-3 deficient mice. Thus, the Tim-3/galectin-9 pathway contributes to but is not essential for MDSC expansion. Future studies should investigate whether Tim-3+ dendritic cells also promote or contribute to MDSC expansion. This will require the use of mice in which Tim-3 is deleted only in T cells or the use of Tim-3−/− × Rag−/− mice as recipients of Tim-3 Tg+ versus Tim-3 Tg− cells. Neither of these strains are currently available.
MDSC are present in normal individuals at a low frequency and, as stated above, accumulate in large numbers in cancer patients and in tumor-bearing mice. Expansion of MDSC has also been observed after exposure to bacterial (24
), parasitic (6
) , viral (34
) antigens, and after traumatic stress (35
). Therefore MDSC may be part of a feedback mechanism induced to prevent damage caused by prolonged or excessive inflammation mediated by Th1 cells. Thus, the Tim-3/galectin-9 pathway regulates proinflammatory Th1 cells by two mechanisms: directly by triggering cell death in Th1 T cells and indirectly through a “cross-talk” with the myeloid compartment. The value of this pathway is further underscored by the findings that Tim-3 expression is dysregulated in human autoimmune and infectious diseases. In the case of multiple sclerosis, TIM-3 expression on T cells infiltrating the CNS was reduced (36
) and in HIV-infected patients, TIM-3 was found to be over-expressed on “exhausted” T cells (37
). However, whether MDSC are affected in these disease conditions was not evaluated. It is possible that part of the exhausted phenotype observed in chronic viral infections is due to an expansion of MDSC. The identification of this mechanism not only illustrates the importance of the dynamic interplay between adaptive and innate immunity in the regulation of effector T cell responses, but also opens new avenues of investigation in MDSC biology. Since the Tim-3/galectin-9 pathway regulates IFN-γ-producing Th1 cells, which are central components in the immune response to infection, autoimmunity and cancer, targeting this pathway may prove beneficial in multiple disease states.