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IFN-γ plays a central role in anti-tumor immunity. Tim-3 is expressed on IFN-γ-producing Th1 cells; upon interaction with its ligand, galectin-9, it terminates Th1 immunity. Here, we show that transgenic over-expression of Tim-3 on T cells results in an increase in CD11b+Ly-6G+ cells and inhibition of immune responses. Molecular characterization of CD11b+Ly-6G+ cells reveals a phenotype consistent with granulocytic myeloid-derived suppressor cells (MDSC). Accordingly, we find that modulation of the Tim-3/galectin-9 pathway impacts on tumor growth. Similarly, overexpression of Tim-3 ligand, galectin-9, results in an increase in CD11b+Ly-6G+ cells and inhibition of immune responses. Loss of Tim-3 restores normal levels of CD11b+Ly-6G+ cells and normal immune responses in galectin-9 transgenic mice. Our data uncover a novel mechanism by which the Tim-3/galectin-9 pathway regulates immune responses and identifies this pathway as a therapeutic target in diseases where MDSC are disadvantageous.
This is an author-produced version of a manuscript accepted for publication in The Journal of Immunology (The JI). The American Association of Immunologists, Inc. (AAI), publisher of The JI, holds the copyright to this manuscript. This manuscript has not yet been copyedited or subjected to editorial proofreading by The JI; hence it may differ from the final version published in The JI (online and in print). AAI (The JI) is not liable for errors or omissions in this author-produced version of the manuscript or in any version derived from it by the United States National Institutes of Health or any other third party. The final, citable version of record can be found at www.jimmunol.org.
After encountering specific antigen, naïve T cells activate, expand and differentiate into various effector T cell subsets such as, Th1, Th2 and Th17 that are characterized by distinct patterns of cytokine secretion. These T cell subsets have specific effector functions and recruit different cell types as a result of the cytokines they produce. Th1 cells produce IFN-γ, Th2 cells produce IL-4, IL-5, IL-13 and Th17 cells produce IL-17, IL-21, and IL-22. Th1 cells are critical in protection against intracellular pathogens and have long been associated with the pathogenesis of many organ-specific autoimmune diseases as IFN-γ was found to be present in the target organ during the peak of disease and Th1 cells were shown to adoptively transfer disease (reviewed in (1)). Moreover, IFN-γ plays a central role in the immune response to tumors (reviewed in (2)). Although it is clear that IFN-γ can be immune stimulatory in that it upregulates the expression of MHC and activates macrophages and neutrophils, IFN-γ has also been suggested to have immunosuppressive properties. Indeed, IFN-γ−/− and IFN-γ R−/− mice develop more severe organ-specific autoimmunity than wild type mice (3–5) suggesting that IFN-γ is in some way involved in suppressing immune responses. Indeed, IFN-γ has been shown to be involved in the induction of myeloid cells with suppressive properties (6–12), but how IFN-γ or IFN-γ-producing Th1 cells induce myeloid cells with suppressive function is not well understood.
To better identify and characterize Th1 cells in vivo, we undertook an antibody screening approach and identified cell surface molecules that are selectively expressed on Th1 but not Th2 cells. Tim-3 is a member of the recently discovered Tim (T cell Immunoglobulin and mucin domain) family that is specifically expressed on IFN-γ-secreting Th1 cells but not Th2 cells (13) and is constitutively expressed on dendritic cells but not on peripheral macrophages (CD11b+CD11c− cells) (14). Tim-3 and its ligand, galectin-9, regulate Th1 immunity by directly triggering cell death in IFN-γ-secreting Th1 cells (15). Tim-3/Tim-3 ligand interactions also play an important role in mediating immune tolerance as mice treated with Tim-3 Ig fusion protein and Tim-3−/− mice cannot be tolerized by high dose aqueous antigen (16). Similarly, Tim-3 Ig-treated mice and Tim-3−/− mice are also refractory to allograft tolerance induced by donor specific transfusion and anti-CD40 ligand antibody or CTLA-Ig treatment (17). Based on these data, it is clear that Tim-3/Tim3-ligand interactions are important in the suppression of immune responses induced by different tolerizing regimens, however the mechanism is not clear. To study more precisely the mechanism by which Tim-3 on T cells induces tolerance in the immune system, we generated mouse strains that overexpress either Tim-3 or galectin-9 (18).
In this study, we elucidate a new mechanism by which the Tim-3/galectin-9 pathway regulates Th1 immunity. We find that Tim-3 Tg mice exhibit depressed T cell responses in comparison to wild-type control littermates and that Tim-3 Tg mice contain a higher frequency of CD11b+Ly-6G+ cells that exhibit a phenotype and morphology consistent with that described for granulocytic myeloid-derived suppressor cells (MDSC). Similarly, overexpression of the Tim-3 ligand, Galectin-9 is associated with an increase in CD11b+Ly-6G+ cells and loss of Tim-3 reverses this expansion and restores normal immune responses. Thus, the Tim-3/galectin-9 pathway regulates Th1 immune responses through at least two mechanisms; directly, by triggering cell death in Th1 cells and, indirectly, through the expansion of suppressive CD11b+Ly-6G+ cells.
For the generation of Tim-3 transgenic mice the full-length cDNA of Tim-3 (Balb/c strain) was cloned into the human CD2 expression cassette (19) and the construct micro-injected directly into C57BL/6 oocytes. Mice expressing galectin-9 under the control of the β-actin promoter (18) and Tim-3−/− (16, 17) mice are on the Balb/c background and were described previously. All animals were housed according to the guidelines established by the Harvard Committee on Animals.
Single cell suspensions from thymus, lymph node or spleen were prepared and stained with the indicated antibodies. Spleens were subjected to digestion with collagenase D (Roche). Anti-CD4, CD8, Tim-3 (8B.2C12), CD11b, CD44, CD45, CD62L, F4/80, Ly-6G, Ly-6C antibodies were purchased from Ebiosciences and BD Biosciences. Unless otherwise noted antibody clone 1A8 was used for Ly-6G staining. All flow cytometry data were collected on a BD FACS Calibur or LSRII (BD Biosciences) and analyzed with FlowJo Software (Tree Star).
Lymphocytes were cultured in triplicate with soluble anti-CD3 in the presence of irradiated antigen presenting cells. For some experiments, CD11b+CD11c− cells were sorted by flow cytometry and used as antigen presenting cells. At 48 h, supernatants were collected for the measurement of cytokines and plates pulsed with [3H]Thymidine and harvested 16 hours later. Cytokines were measured from culture supernatants by either ELISA or cytometric bead array (CBA) (BD Biosciences).
EL4 thymoma cells (from C57BL/6) and 4T1 mammary adenocarcinoma (from Balb/c) were cultured at 37°C under 10% CO2 in RPMI 1640 medium supplemented with 10% FCS, penicillin, streptomycin and 1 mM pyruvate. Wild type or Tim-3Tg C57BL/6 mice were injected with 5×105-1×106 EL4 cells within the right flank. 1×105 4T1 cells were injected into mammary tissue of Tim-3−/− or wild type Balb/c mice. Tumor surface was monitored in two dimensions three times per week using a caliper. In some experiments, mice were treated with 100 µg of either anti-Tim-3 antibody (clone 5D12), anti-Ly-6G antibody (clone 1A8) or isotype controls intraperitoneally.
6 week old wild-type or Tim-3 transgenic littermates were injected subcutaneously with 100 µg of MOG 35–55 peptide (MEVGWYRSPFSRVVHLYRNGK) emulsified in CFA (Difco) supplemented with 4 µg ml−1 Mycobacterium tuberculosis and injected twice intravenously with 100 ng of pertussis toxin (List Biological Laboratories). Clinical assessment of EAE was as follows: 0, no disease; 1, decreased tail tone; 2, hindlimb paresis; 3, complete hindlimb paralysis; 4, forelimb and hindlimb paralysis; 5, moribund state.
EAE was induced in Tim-3 Tg mice and wildtype littermates by immunization with 100 µg of MOG 35–55 emulsified in complete Freund’s adjuvant (Difco) supplemented with 4 µg ml−1 Mycobacterium tuberculosis and injected twice intravenously with 100 ng of pertussis toxin (List Biological Laboratories). At different stages of disease, mice were sacrificed and CNS mononuclear cells obtained by percoll gradient centrifugation of collagenase digested CNS tissue (brain and spinal cord). Cells were then stained with antibodies to CD11b, CD45, Ly-6G and F4/80 and analyzed on a BD FACSCalibur.
Mice were immunized subcutaneously with 100 µg of TNP-OVA in CFA. On day 10, draining lymph nodes were harvested and restimulated with TNP-OVA. Proliferation and cytokine production were measured as described above.
1.5–2×106 sorted CD4+ Tim-3 Tg+ or Tg− cells were injected intravenously into 6 week old Rag1−/− C57BL/6 mice. On day 35–40 post-transfer, spleens were harvested and digested with collagenase D (Roche) prior to analysis by flow cytometry.
RNA was isolated using Qiagen RNeasy and used for quantitative PCR. The forward and reverse primers for galectin-9 amplification are as follows: 5’Gal9: 5’-GTTGTCCGAAACACTCAGAT-3’; 3’Gal-9: 5’-ATATGATCCACACCGAGAAG-3’; probe:5’-CAGGAAGAGCGAAGTCTGCT-3’. Gene expression was normalized to the housekeeping gene GAPDH.
We generated a Tim-3 transgenic (Tim-3 Tg) mouse by expressing the full-length Tim-3 cDNA (Balb/c isoform) under the control of the human CD2 promoter (19) on the C57BL/6 genetic background. In these mice, we are able to track Tim-3 transgene positive cells with an antibody specific for the Balb/c isoform of Tim-3 (clone 8B.2C12). Tim-3 Tg mice are viable, fertile and do not exhibit any gross alterations in the size of lymphoid organs. In the thymus, the Tim-3 transgene is expressed at the double negative (DN) stage and maintained through the double positive (DP) and single positive (SP) stages of thymocyte development. Interestingly, the Tim-3 transgene is expressed in only 30–40% of CD4+SP and CD8+SP thymocytes (Fig. 1A). Analysis of thymic development showed a small decrease in the frequency of Tim-3 Tg+ DN thymocytes but no statistically significant difference in the frequency of Tim-3 Tg+ DP, CD4SP and CD8SP thymocytes when compared to Tim-3 Tg− thymocytes (data not shown).
In the periphery, we observed no alterations in the frequency or numbers of T or B cells in Tim-3 Tg mice compared to littermate controls (data not shown). As observed in the thymus, only 30–40% of peripheral CD4+ T and CD8+ T cells express the Tim-3 transgene (Fig. 1A). We next characterized the effector/memory phenotype of peripheral CD4+ T cells and found no major differences in the expression of either CD62L and CD44 in the whole CD4+ T cell compartment of wild type and Tim-3 Tg mice (Fig. 1B). However, when CD4+ T cells from Tim-3 transgenic mice were segregated into Tim-3 Tg+ and Tim-3 Tg− populations, the Tim-3 Tg+ T cell population contained significantly fewer effector/memory (CD44high and CD62Llow) T cells compared to the Tim-3 Tg− population (Fig. 1B), suggesting that Tim-3 expression on T cells controls effector/memory cell generation. The same trend was observed with CD44 but not CD62L expression on CD8+ T cells (data not shown). Importantly, the CD4+Tim-3 Tg− T cell compartment was not affected in that the ratio of effector/memory (CD44high or CD62Llow) to naïve (CD44low or CD62Lhigh) cells in these cells was similar to that of CD4+ T cells from wild type littermate controls (Fig. 1B).
To determine the effect of Tim-3 overexpression on a limited number of T cells on the total T cell response, we stimulated total splenocytes from Tim-3 transgenic mice with anti-CD3. Although Tim-3 is expressed on a limited number of T cells, T cell proliferation was decreased and IFN-γ production was reduced to background levels when compared to cultures from wild type littermates (Fig. 1C). These data suggested the presence of a dominant factor(s) in Tim-3 Tg mice that suppresses T cell responses.
As antigen presenting cells (APC) are an integral component of T cell proliferation we examined the APC compartment in Tim-3 Tg mice for alterations. We found no significant difference in the frequency or number of CD11c+ dendritic cells or CD19+ B cells (data not shown). However, we did find that the CD11b+ population in Tim-3 Tg mice was double that of wild type littermates (Fig. 1D). As the CD11b+ population contains many cell types including monocyte/macrophages and granulocytes, we further characterized the phenotype of the expanded CD11b+ cells in Tim-3 Tg mice by staining for F4/80, a marker for monocyte/macrophages and eosinophils and Gr-1, a marker for inflammatory monocytes and granulocytes. We found that the proportion of CD11b+ cells that are Gr-1+ and F4/80low is increased in Tim-3 Tg mice relative to wild type littermates (Fig. 1D). Absolute numbers of CD11b+Gr-1+ are increased approximately 2–3 fold in Tim-3 Tg mice relative to wild type littermates (0.65–2.2×106 in wild type and 1.4–6.7×106 in Tim-3 Tg). To test the functional consequences of an increase in this population for T cell activation, we compared the ability of_the total CD11b+ population from Tim-3 Tg and wild type mice to stimulate T cell proliferation and found that the total CD11b+ population from Tim-3 Tg mice suppressed both proliferation and IFN-γ secretion from wild type CD4+ T cells (Fig. 1E). Collectively, these data suggest that the expansion of CD11b+Gr-1+F4/80low cells is associated with dampened T cell immunity in Tim-3 Tg mice.
CD11b+Gr-1+ cells have been shown to arise in tumor-bearing mice and their presence is associated with poor clinical outcome (reviewed in (20–22)). Accordingly, we examined the expansion of CD11b+Gr-1+ cells in the spleens of wild type and Tim-3 Tg mice implanted with the T cell lymphoma, EL-4. We found that Tim-3 Tg mice exhibited more CD11b+Gr-1+ cells in the spleen compared to wild type littermates (Fig. 2A). In keeping with the higher expansion of CD11b+Gr-1+ cells in Tim-3 Tg mice, EL-4 tumors grew more rapidly in Tim-3 Tg mice (Fig. 2A). Moreover, treatment of EL-4 tumor bearing mice with anti-Tim-3 antibody resulted in delayed tumor progression coincident with lower frequency of CD11b+Gr-1+ cells (Fig. 2B). As we have Tim-3−/− mice on the Balb/c background, we examined the growth of a Balb/c tumor, 4T1 mammary adenocarcinoma, in wild type and Tim-3−/− deficient mice and found that tumor progression was significantly delayed in Tim-3−/− deficient mice and this was again coincident with lower frequency of CD11b+Gr-1+ cells (Fig. 2C).
Because the Gr-1 antigen consists of epitopes from both Ly-6G and Ly-6C, we further examined the expression of these two molecules with specific antibodies and found that the proportion of CD11b+ cells that are Ly-6G+ and Ly-6Clow is higher in Tim-3 Tg mice relative to wild type littermates (Supplementary Fig. 1). Thus, Tim-3 Tg mice have more CD11b+ cells that are Ly-6G+F4/80lowLy-6Clow. To further examine the nature of the CD11b+ cells that are expanded in Tim-3 Tg mice, we isolated the CD11b+Ly-6G+F4/80low cells and compared their morphology to that of CD11b+Ly-6G−F4/80high cells. We found that CD11b+Ly-6G+F4/80low cells contained cells with complex nuclear morphology including immature cells with ring-shaped nuclei with a wide cytoplasmic center (Supplementary Fig. 2). This nuclear morphology has been previously associated with immature myeloid cells that are suppressive (10, 23, 24). In contrast, CD11b+Ly-6G−F4/80high cells consisted of cells with classic monocyte and eosinophil morphology. While the morphology of the CD11b+ Ly-6G+F4/80low cells was similar in wild type and Tim-3 Tg mice, both the frequency of this population and the frequency of immature cells with ring-shaped nuclei within this population was higher in Tim-3 Tg mice than in wild type mice. Furthermore, the gene expression profile of CD11b+Ly-6G+F4/80low cells in both wild type and Tim-3 Tg mice showed striking similarities to that of CD11b+ cells isolated from tumor-bearing mice (9) (Supplementary Fig. 3).
We therefore examined the effect of anti-Tim-3 antibody on the expansion of CD11b+Ly-6G+ cells in a wild type host and found that CD11b+Ly-6G+F4/80low cells expand less in mice treated with anti-Tim-3 relative to mice treated with isotype control (Supplementary Fig. 4). This is in keeping with our observation that CD11b+Gr-1+ cells expand less in anti-Tim-3 treated tumor-bearing mice (Fig. 2B). We therefore tested the effect of depletion of Ly-6G+ cells in EL-4 tumor bearing mice and found that mice treated with anti-Ly-6G antibody exhibited delayed tumor growth relative to mice treated with isotype control antibody (Fig. 2C). Collectively these data suggest that interference with Tim-3 signaling, either in Tim-3 transgenic mice, mice treated with anti-Tim-3 antibody or in Tim-3−/− mice, impacts on tumor progression by altering the expansion of CD11b+Ly-6G+F4/80low cells which then negatively impacts on T cell activation.
Given that Tim-3 Tg mice exhibit an increase in CD11b+Ly-6G+F4/80low cells and defects in polyclonal T cell responses in vitro, we next examined how the generation of autopathogenic immune responses in vivo would be affected in Tim-3 Tg mice. We therefore immunized Tim-3 Tg mice for the development of Experimental autoimmune encephalomyelitis (EAE), a model of central nervous system (CNS) autoimmunity in which myelin-specific effector T cells play a central role in disease induction. We found that Tim-3 Tg mice are protected against EAE in that the incidence of clinical disease in Tim-3 Tg mice is a third of that of wild type littermates (Fig. 3A and B, Table 1). Given the expansion of CD11b+ Ly-6G+ F4/80low cells we had observed in the periphery of Tim-3 Tg mice, we hypothesized that the resistance to autoimmune disease in Tim-3 Tg mice was due to the infiltration of these cells from the periphery into the CNS, where they could suppress effector T cell-mediated inflammation and demyelination. We therefore examined the frequency of CD11b+ Ly-6G+ F4/80low cells infiltrating the CNS in both wild type and Tim-3 Tg mice with and without disease. We found that the presence of CD11b+Ly-6G+F4/80low cells in the CNS infiltrate of mice immunized for EAE correlated with the absence of clinical disease and that the frequency of these cells in the CNS infiltrate drops dramatically in diseased mice (Fig. 3C and D). It is important to note that since Tim-3 Tg mice have more CD11b+Ly-6G+F4/80low cells, more Tim-3 Tg than wild type mice were disease-free (n=6 vs n=3, respectively, Fig. 3D). Collectively these data support a model where Tim-3 inhibits the generation of pathogenic T cells directly by promoting the termination of Th1 cells and also indirectly by promoting the expansion of CD11b+Ly-6G+F4/80low cells that in turn suppress the generation of autopathogenic T cell responses. Given that the majority of T cells in Tim-3 Tg mice do not express the Tim-3 transgene and thus are not subject to galectin-9-mediated regulation, these data further suggest that the role of Tim-3 in suppressing autopathogenic T cell responses through the promotion of CD11b+Ly-6G+F4/80low myeloid cells is dominant.
To address whether Tim-3 expression on T cells triggers the expansion of CD11b+Ly-6G+F4/80low cells in vivo, we isolated CD4+ Tim-3 Tg+ and CD4+ Tim-3 Tg− cells from Tim-3 Tg mice and transferred them into Rag1−/− recipients, which lack T and B cells but have an intact myeloid compartment. After 4–6 weeks, the extent of reconstitution as determined by splenic cellularity was not different between recipients of CD4+ Tim-3 Tg+ and CD4+ Tim-3 Tg− T cells (Fig. 4A); however, Rag1−/− mice reconstituted with CD4+ Tim-3 Tg+ T cells contained significantly higher percentages of CD11b+ cells (Fig. 4B). When we further characterized the CD11b+ cells present in the Rag1−/− reconstituted mice, we found that the mice reconstituted with Tim-3 Tg+ T cells had a significantly higher frequency of CD11b+ cells that were Ly-6G+ and F4/80low (Fig. 4C). Consistent with this observation, the absolute number of CD11b+Ly-6G+ F4/80low is higher in the Rag1−/− mice reconstituted with Tim-3 Tg+ T cells compared to Tim-3 Tg− T cell reconstituted mice (Fig. 4D). These data directly show that increased expression of Tim-3 on T cells promotes expansion of CD11b+ Ly-6G+F4/80low cells.
Given that galectin-9 is a ligand for Tim-3, we next examined whether galectin-9 is similarly involved in the expansion of CD11b+Ly-6G+F4/80low cells that suppress T cell responses. If galectin-9 is involved, then overexpression of galectin-9 should phenocopy overexpression of Tim-3. We therefore examined the ability of T cells from galectin-9 transgenic (Gal-9 Tg) mice to proliferate and produce cytokines. Although proliferation in response to anti-CD3 was not different, we observed a significant decrease in IFN-γ production with a concomitant increase in IL-10 and IL-4 production in cells from Gal-9 Tg mice compared to wild type littermate controls (Fig. 5A). We next addressed the in vivo relevance of these findings by immunizing Gal-9 Tg mice and wild type littermates with TNP-OVA and found a dramatic decrease in the ability of cells from immunized Gal-9 Tg mice to proliferate and produce IFN-γ upon in vitro reactivation (Fig. 5B). IL-4 and IL-10 were not detected since the mice were immunized with CFA. Similar to Tim-3 Tg mice, in Gal-9 Tg immunized mice, there was a significant decrease in the CD4+CD62Llow effector/memory T cell population (Fig. 5C). Thus, Galectin-9 transgenic mice appear to phenocopy Tim-3 Tg mice in that there is a marked decrease in the ability of Gal-9 Tg mice to prime Th1 immune responses and generate effector/memory cells.
Given our observation that defective T cell responses are associated with an expansion of CD11b+Ly-6G+F4/80low cells in Tim-3 Tg mice, we examined whether these cells are similarly expanded in Gal-9 Tg mice. Indeed, we observed a significant increase in the CD11b+ myeloid population in Gal-9 Tg mice but no significant differences in the numbers/frequency of CD11c+ dendritic cells or CD19+ B cells (Fig. 5D and data not shown). Further characterization of the CD11b+ cells from Gal-9 Tg mice revealed an increase in the proportion of CD11b+Ly-6G+F4/80low cells in Gal-9 Tg mice (Fig. 5D). Altogether these data demonstrate a significant expansion of CD11b+Ly-6G+F4/80low cells in mice overexpressing galectin-9 and suggest an important role for galectin-9 in the promotion of these cells.
To determine if the expansion of CD11b+Ly-6G+F4/80low cells may be responsible for the defect in priming Th1 immune responses in Gal-9 Tg mice, we compared the ability of the total CD11b+ population obtained from Gal-9 Tg mice and wild type littermates to activate wild type T cells. We observed a marked decrease in proliferative responses with CD11b+ cells from Gal-9 Tg mice (Fig. 5E). This was accompanied by an almost total absence of IFN-γ production with a concomitant increase in IL-4 and IL-10 production (Fig. 5E). Collectively, these data suggest that the expansion of CD11b+Ly-6G+F4/80low cells within the total CD11b+ population could be responsible for the defect in productive Th1 immune responses observed in Gal-9 Tg mice in response to immunization.
To understand how transgenic expression of both Tim-3 and its ligand, galectin-9, could independently result in the expansion of CD11b+Ly-6G+F4/80low cells, we examined the expression of galectin-9 by CD11b+Ly-6G+ cells and found that they naturally express more galectin-9 than CD11b+Ly-6G− cells and that this difference is further augmented by activation of these cells with the Th1 cytokine IFN-γ (Fig. 6). The higher expression of galectin-9 on CD11b+Ly-6G+ cells provides a basis for the ready interaction of Tim-3 expressing T cells with galectin-9 for the expansion of these cells. To determine whether a direct interaction of Tim-3 with galectin-9 is responsible for the expansion of CD11b+Ly-6G+F4/80low cells that we observe, we crossed Gal-9 Tg mice with Tim-3−/− mice. Comparison of Gal-9 Tg, wild type, and Gal-9 Tg × Tim-3−/− mice revealed that the increased frequency of CD11b+ cells observed in Gal-9 transgenic mice was restored to wild type levels in Gal-9 Tg × Tim-3−/− mice (Fig. 7A). Further characterization of the CD11b+ cells obtained from the different mouse strains demonstrated that the proportion of CD11b+ cells that are CD11b+Ly-6G+F4/80low is also restored to wild type levels in Gal-9 Tg × Tim-3−/− mice, thereby confirming that the expansion of these cells in Gal-9 Tg mice is dependent on the interaction with Tim-3 (Fig. 7A). Moreover, the CD11b+ cells from Gal-9 Tg × Tim-3−/− mice were equally able to stimulate proliferation and IFN-γ production from wild type CD4+ T cells as CD11b+ cells taken from wild type mice (Fig. 7B). Similarly, the priming of Th1 immune responses was restored to wild type levels in Gal-9 Tg × Tim-3−/− mice immunized with TNP-OVA (Fig. 7C). Lastly, the defect in the generation of CD62Llow effector/memory cells observed in Gal-9 Tg mice following immunization was also corrected in immunized Gal-9 Tg × Tim-3−/− mice such that the Gal-9 Tg × Tim-3−/− mice had close to wild type levels of effector/memory T cells (Fig. 7D).
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, 26). 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+Ly-6G+F4/80lowLy-6Clow cells. Interestingly, this cell surface phenotype is consistent with that described for granulocytic or PMN-like myeloid-derived suppressor cells (MDSC) (10, 27). In addition, our morphological analysis of CD11b+Ly-6G+F4/80low 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 (20–22)). 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+Ly-6G−Ly-6Chi and granulocytic (CD11b+Ly-6G+Ly-6Clow) (10, 27). 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, 27). 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 (6–10). 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+Ly-6G+F4/80low 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+Ly-6G+F4/80low 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, 29) and to activate MDSC both in vitro and in vivo (30). Moreover, IL-1R−/− mice exhibit a defect in MDSC accumulation (31). The expression profile of the CD11b+Ly-6G+F4/80low 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+Gr-1+ 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, 33), parasitic (6, 7) , 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.
The authors would like to acknowledge Jenna Sullivan for technical assistance.
This work was supported by grants from the National Institutes of Health (VKK and ACA), the National Multiple Sclerosis Society (VKK and VD), the Juvenile Diabetes Research Foundation Center for Immunological Tolerance at Harvard (VKK and JK), and EMBO (LA). VKK is a recipient of the Javits Neuroscience Investigator Award from the National Institutes of Health.
Disclosure of Conflicts of Interest