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Tumor-induced immunosuppression plays a key role in tumor evasion of the immune system. A key cell population recognized as myeloid-derived suppressor cells (MDSC) contributes and helps orchestrate this immunosuppression. MDSC can interact with T cells, macrophages, and NK cells, to create an environment favorable for tumor progression. In various tumor models their presence at high levels has been reported in the bone marrow, blood, spleen, and tumor. We report for the first time that MDSC accumulate and home to the liver in addition to the other organs. Liver MDSC suppress T cells and accumulate to levels comparable to splenic MDSC. Additionally, hematopoiesis in the liver contributes to the dramatic expansion of MDSC in this organ. Furthermore, MDSC in the liver interact with macrophages, also known as Kupffer cells, and cause their up-regulation of PD-L1, a negative T cell costimulatory molecule. The liver is thus an organ where MDSC accumulate and can contribute to immunosuppression directly and indirectly. MDSC play a role in various pathological states in addition to cancer, and these results contribute to our understanding of their biology and interactions with immune-related cells.
Tumor-induced immunosuppression is accepted as a key mechanism by which tumors evade the immune system. This is a critical capability which tumors gain in the last stage of the immunoediting process(1). Nearly twenty years ago, we described hematopoietic changes in tumor-bearing mice involving myeloid cells which accumulate in the spleen due to tumor-derived factors (2). We reported that these CD11b+(MAC-1) cells down-regulate polyclonal and antigen-specific T and B cell responses (3). These cells, now described as CD11b+GR-1+ myeloid-derived suppressor cells (MDSC), suppress T cells in various cancer models (4, 5) and patients (6). These cells have also been described in other pathological states, including including inflammatory bowel disease (7), traumatic stress (8), burns (9), infection (10), and transplantation(11).
Besides suppressing CD4+ and CD8+ T cells, MDSC interact with other immune regulatory cells (12), and block anti-tumor immunity. They have been found to block NK cell cytotoxicity (13), they modulate macrophages to an immunosuppressive M2 phenotype(14), and can induce the development of T regulatory cells (Treg)(15). We discovered that MDSC home to and accumulate in high numbers in the liver. Furthermore, the dramatic increase in systemic levels of MDSC in tumor bearers is in part explained by extramedullary hematopoiesis, as increased levels of hematopoietic progenitors exist in the spleen and liver. We also report for the first time that in tumor-bearers’ the liver plays a critical role in immunosuppression by accumulating MDSC which interact with liver macrophages, or Kupffer cells, and inducing high levels of the negative T cell costimulatory molecule PD-L1 on their surface.
BALB/c mice (H-2d) were bred in our animal facility at the University of Miami according to NIH guidelines. C57BL/6 mice were purchased from the Jackson Laboratory. DO11.10 transgenic mice were kindly provided by Dr. Becky Adkins (University of Miami , Miami, FL). The DA-3 mammary tumor cells were maintained as previously described (2). The 4T1 cell line was kindly provided by Dr. Fred Miller (Wayne State University, Detroit, MI). B16.F10 and LLC cells were purchased from ATCC (Manassas, VA). Tumor cells (1×106 DA-3, 1×104 4T1, 2.5×105 B16 or LLC) were injected s.c., and then 4-5 wk tumor-bearing animals were used for indicated studies. Recombinant murine GM-CSF (R&D Systems, Minneapolis, MN) was injected i.p. in 0.9% saline. For the in vivo PD-L1 experiment, mice inoculated with DA-3 cells s.c. were administered i.p. either saline or 150μg anti-PD-L1 (Biolegend, San Diego, CA) every 3-4 days, while tracking tumor development.
Spleens were mashed through 70μM cell strainers (BD Biosciences, San Jose, CA) to obtain single-cell suspensions. Liver leukocytes were harvested by removing livers, chopping them to pieces, and resuspending in 4.5mL HBSS and 350μl of digestion enzyme mixture provided in a protocol by Dr. Alan B. Frey (New York University, NY) (16). The stock digestion enzyme mixture consisted of 5mg/ml Collagenase I, 5mg/ml Collagenase IV, 2.5mg/ml Hyaluronidase V (Sigma-Aldrich, St. Louis, MO), and 1mg/ml DNase I (Roche, Indianapolis, IN). Tissue/enzyme mixture was placed in a 37°C shaker for 30 minutes, then poured through 70μM cell strainers (BD Biosciences). To remove hepatocytes, cells were resuspended in 1.2ml RPMI, and 2.8ml of 50% Histodenz (Sigma-Aldrich) solution in PBS, and underlayed in tubes containing 2.0ml of RPMI. Mixture was centrifuged for 20 minutes at 1500×g, 5°C. Interphase cells were removed and washed using RPMI. Purification of MDSC/Kupffer cells were performed by staining cells using anti-GR-1 (RB6-8C5, BD Biosciences) or anti-F4/80 (Invitrogen), respectively, followed by magnetic antibody cell separation (MACS) using goat anti-rat microbeads (Miltenyi Biotec, Auburn, CA). For negative selection of MDSC, cells were stained with biotin conjugated anti-CD4, anti-CD8, and anti-CD45 from BD Biosciences, followed by separation using BD IMag™ SAv particles (BD Biosciences).
Cells were labeled with 2μM DiR (Invitrogen)(17). In other experiments, 40μg of anti-GR-1.APC-Cy™7 (BD Biosciences) was injected i.v.. Mice were imaged and analyzed using a optical bioimager (IVIS 200; Xenogen, Hopkintown, MA) with Ex/Em of 745nm/820nm for DiR or 640nm/800nm for APC-Cy™7.
Paraffin embedding, sectioning, and staining with H&E was performed at the Comparative Pathology laboratory (University of Miami). To analyze purified MDSC, 1×106 cells were dried onto slides, fixed in methanol, and stained using Harleco Hemcolor (EMD Chemicals, Gibbstown, NJ). Immunohistochemistry was performed by fixing cryo-preserved liver sections in cold acetone for 5 minutes, allowing to air-dry, and after rehydrating with PBS and blocking with antibody diluent (BD Biosciences) sections were stained with anti-GR-1.APC and anti-CD11b.FITC. Sections were then washed thoroughly and cover-slipped with VectaShield DAPI mounting medium (Vector Laboratories, Berlingame, CA). Slides were analyzed and photographed using a Zeiss LSM510/UV inverted confocal microscope at the Analytical Imaging Core Facility (University of Miami).
Cells were Fc blocked and antibody labeled in FACS buffer (PBS, 0.5%BSA, 0.1% sodium azide), then analyzed on an LSR II(BD Biosciences). Antibodies used: anti-GR-1. APC, anti-CD11b.PE, anti-PD-L1.PE, anti-CD4.PE-Cy5 from BD Biosciences, anti-F4/80.FITC from Invitrogen, and anti-B7-H4.PE from eBioscience (San Diego, CA).
Cell suspensions were diluted to 2×105cells/mL in IMDM medium with 2% heat-inactivated FCS for mouse myeloid colony-forming cells (StemCell Technologies, Vancouver, CA) and 10xpenicillin/streptomycin. 0.3mL of cell solution was then mixed with 3mL MethoCult GF M3434 for CFU-GEMM clonogenic potential, or MethoCult M3001 for CFU-GM clonogenic potential (StemCell Technologies). Cells were then plated in duplicate at 1mL/well in 6 well dishes and incubated for 11 days before scoring the number of colonies.
DO11.10 splenocytes were labeled with 2.5μM CFSE for 10 minutes at 37°C. Labeled splenocytes were plated at 2×105 cells per 96 flat-bottom well, in HL-1 media (BioWhittaker, Walkersville, MD) containing penicillin/streptomycin, glutamax, and 2-ME. Ovalbumin (OVA, EMD/Calbiochem) was added at 15μg/mL. MDSC purified by MACS were added at indicated concentrations. After 5 days, cells labeled with anti-CD4, were analyzed for CFSE dilution. For Kupffer assays, DO11.10 splenocytes were pre-activated with 50μg/mL OVA for 8 days. T cells were purified using MACS CD90 beads and co-cultured (2×105) with Kupffer cells (5×104), or splenic dendritic cells purified using MACS CD11c beads, in the presence of 5ug/mL OVA. F(ab’)2 fragments of anti-PD-L1 (Biolegend, San Diego, CA) or isotype were made using F(ab’)2 micro preparation kit (Pierce, Rockford, IL), and added to cultures at 10μg/mL. IFN-γ was analyzed in 4-day cultures by ELISA (BD Biosciences).
MDSC(CD11b+GR-1+) have been reported at high levels in spleen, bone marrow, and blood of tumor-bearing mice, as well as infiltrating primary tumors. To investigate MDSC homing, splenic MDSC were isolated from BALB/c mice harboring mammary DA-3 tumors, and labeled with a near-infrared dye(DiR). MDSC were then injected (4×106) i.v. into either normal or tumor-bearing mice. After 24 hours, the mice were imaged using a bioimager, and displayed the highest intensity in the spleen and livers of both tumor-bearing and normal mice (Fig.1A). MDSC have not been previously described to home to the liver, however it is also possible that dead cells or released dye accumulate for removal by the liver(17). To determine whether MDSC actually accumulate in the liver of tumor-bearing mice, normal BALB/c mice or mice harboring a DA-3 tumor were injected i.v. with an antibody against GR-1(Anti-GR-1. APC-Cy™7), and imaged using a bioimager 20 minutes later. High intensity of labeling was found in tumor bearers relative to normal mice, with 1.8 fold, 7.3 fold, and 11.5 fold higher signals from the liver, spleen, and bones, respectively (Fig.1B). Some signal from the liver is due to removal of antibody, however the signal from livers of tumor-bearing mice was higher than that of normal liver.
To analyze further whether MDSC accumulate in livers of tumor-bearing mice, livers from normal mice or DA-3 tumor-bearers were isolated, fixed in formalin, and embedded in paraffin. In livers from tumor-bearers large nests of leukocytes can be detected peri-vascularly as well as throughout the liver parenchyma (Fig.1C). Frozen sectioning of liver tissue reveals the nuclei of these cells, which are characteristic of MDSC that are a heterogeneous population of cells including immature myeloid cells with rounded nuclei, bands, and macrophages (Fig.2A). Liver histology was also performed on livers from other tumor models, and the same nest pattern was observed in the livers of 4T1 tumor-bearing mice (Fig.2B). While the DA-3 model is not metastatic to the liver, the 4T1 model does metastasize to the liver, and interestingly some metastatic nodules were surrounded by MDSC (Fig.2B). To further characterize these nests of leukocytes, immunohistochemistry was performed by staining cryo-preserved sections using antibodies to GR-1 and CD11b. Sections of tumor-bearers’ livers showed clusters of intense staining for these two markers, while sections of normal livers showed no co-localized staining (Fig. 2C). MDSC were then purified from the liver and spleens of 4T1 tumor-bearers and stained on slides for comparison, and were similar histologically (Fig.2D).
Systemic levels of MDSC have been correlated to tumor burden, likely due to the levels of tumor-derived factors shown to stimulate their production and prevent their maturation(2). To investigate how levels of MDSC in the liver compare to the spleen, paired livers and spleens from tumor-bearing and normal animals were harvested, and cells double stained for CD11b and GR-1, and analyzed by flow cytometry. Two BALB/c tumor models, DA-3 and 4T1, and two C57BL/6 tumor models, B16 melanoma and Lewis lung carcinoma (LLC), were analyzed. In all four tumor models, levels of MDSC in the liver paralleled those in the spleen in a linear fashion (Fig.3A). Normal livers resembled normal spleens, with low levels of MDSC at 2-5%, while tumor-bearers’ livers resembled tumor-bearers’ spleens (Fig. 3B). A T cell suppression assay was performed using MDSC purified from either the liver or spleen of the same tumor-bearing animal. CFSE labeled splenocytes from DO11.10 transgenic mice, were stimulated with ovalbumin (OVA) in the absence or presence of MDSC from the two organs. Liver MDSC suppression of T cell proliferation was comparable to splenic MDSC suppression, with 80% suppression at a 1:1 ratio of splenocytes to MDSC, as seen by lack of CFSE dilution in DO11.10 CD4+ T cells (Fig.3C).
In Figure 1 we showed that MDSC adoptively transferred into either normal or tumor-bearing mice accumulated in the spleen and liver of both animals. Furthermore, using tumor models which do (4T1) or do not (DA-3) metastasize to the liver, we found that MDSC accumulate in the liver irrespective of liver metastatic disease. However, MDSC accumulation in the spleen and liver could be a result of tumor-derived factors. We reported in the past that tumor production of granulocyte macrophage-colony stimulating factor (GM-CSF) can lead to the hematopoietic changes and immune dysfunction observed in tumor-bearing mice, including the splenic accumulation of MDSC (2, 18). We thus investigated whether the accumulation of MDSC by GM-CSF can lead to liver MDSC accumulation. BALB/c mice were injected i.p. twice daily with 5μg GM-CSF for 3 days, and then spleens and livers were harvested and analyzed for levels of MDSC. While saline injected control mice harbored low levels of MDSC in both the spleen and the liver, GM-CSF injected mice had up-regulated levels of MDSC in both the spleen and liver (Fig. 4A).
Other investigators have previously reported that expansion of myeloid cells in tumor-bearing mice was in part due to either increased hematopoietic progenitors colonizing the spleen, or increased splenic hematopoiesis (19). Furthermore, since nests of leukocytes were detected throughout the liver, we tested the clonogenic activity in livers, spleens, and bone marrow of DA-3 tumor-bearers and normal mice. The number of hematopoietic colonies, whether assaying early progenitors (CFU-GEMM) or more differentiated progenitors (CFU-GM), were greatly increased from all three compartments of tumor-bearing mice relative to those from normal mice (Fig. 4B). Thus, MDSC likely arise from all three sites, contributing to their dramatic accumulation systemically, and specifically in these organs.
Liver macrophages, known as Kupffer cells, are involved in the induction of immunological tolerance (20), and in the resolution of infections and liver pathology. Mechanisms used in the induction of tolerance by these cells include cytokine secretion and expression of inhibitory costimulatory molecules, such as PD-L1(CD274, B7-H1). We investigated the phenotype of F4/80+ Kupffer cells and splenic macrophages in DA-3 tumor-bearing and normal mice for surface expression of two T cell inhibitory costimulatory molecules, PD-L1 and B7-H4 (21). While PD-L1 is constitutively expressed on liver and spleen F4/80+ cells of normal animals, it is twofold higher on Kupffer cells from tumor-bearing mice (Fig.5A). PD-L1 was also higher in Kupffer cells of B16 tumor-bearing mice (data not shown). Furthermore, this is a Kupffer cell specific phenotype, as splenic macrophages expressed the same levels of PD-L1 from either tumor-bearing or normal mice. B7-H4 was expressed at lower levels, but was identical on macrophages from tumor bearers and normal mice (Fig.5A). Since MDSC accumulate in the livers of tumor-bearers, we investigated whether MDSC induce higher Kupffer cell PD-L1 expression. Adoptive transfer of purified splenic MDSC from tumor bearers i.v. into normal BALB/c mice leads to a rapid increase in Kupffer cell surface PD-L1 (at 2 and 5 hrs; Fig.5B), and returns to baseline within 20 hrs, likely due to the absence of a tumor-bearing immune suppressive environment. To investigate whether up-regulation of PD-L1 on Kupffer cells by MDSC was specifically due to MDSC derived from tumor-bearers, or whether MDSC from normal animals if given at the same high numbers can also up-regulate this immunosuppressive molecule, pooled normal or tumor splenic MDSC were adoptively transferred into normal mice. After five hours, the livers were analyzed for levels of MDSC and PD-L1 expression on Kupffer cells. While saline injected mice harbored approximately 3% MDSC within the liver, mice adoptively transferred with either tumor or normal MDSC harbored 14-15% MDSC within the liver (data not shown). PD-L1 was up-regulated on Kupffer cells in the presence of MDSC, irrespective of their origin from either tumor or normal mice (Fig. 5C). It is also important to note that in our tumor models MDSC are negative for both PD-L1 and F4/80 (data not shown).
PD-L1 has been shown to interact with its receptor PD-1, which is up-regulated on activated T cells, and suppress T cell IFN-γ production (22). To confirm this negative costimulatory function we cultured preactivated DO11.10 T cells in the presence of OVA, with normal or tumor-bearers’ Kupffer cells, or normal splenic dendritic cells as a control. In the presence of PD-L1 blocking F(ab’)2 fragments, the secretion of IFN-γ by T cells co-cultured with Kupffer cells can be up-regulated (Fig.6A). Dendritic cells on the other hand efficiently activate T cells and thus induce a higher production of IFN-γ. There is a lower production of IFN-γ in the presence of tumor Kuppfer cells relative to the presence of normal Kupffer cells (~600pg/mL difference). While we would have predicted a bigger difference based on PD-L1 expression, as it turns out Kupffer cell adherence to plastic in vitro causes PD-L1 to completely down-regulate within an hour and then to up-regulate to similar levels after several hours in vitro (data not shown). Although, we cannot selectively down-modulate PD-L1 on Kupffer cells, we investigated whether in vivo blockade of this ligand systemically in tumor-bearers had any effect on tumor growth. DA-3 tumor-bearing mice receiving anti-PD-L1 treatments twice weekly showed slowed tumor growth relative to mice receiving saline (Fig. 6B). As shown by others, breaking the PD-1/PD-L1 axis alone can slow the growth and metastasis of some tumor models (23, 24). However, it has been shown to be markedly more effective in combination with other antitumor immunotherapies (24, 25).
MDSC are now recognized as key players of immunosuppression in cancer and other chronic inflammatory pathologies. Specifically in cancer, they have been found to expand in almost every mouse model (26), and in various human cancers, including melanoma (27), breast (28), lung (29), renal cell carcinoma (30), and hepatocellular carcinoma (31). We and others previously reported that they accumulate due to tumor-derived factors (2), and suppress T cells in a cell contact dependent manner (3). While mechanisms of their suppressive activity, such as arginase expression (32), and their expansion and recruitment by tumor-derived factors, such as GM-CSF (2, 18, 33), urokinase plasminogen activator (uPA) (34), and stem cell factor (35), are being uncovered, much of their biology remains to be elucidated. Since MDSC accumulation has been reported in the spleen, bone marrow, blood, and within the tumor, we set out to determine where MDSC preferentially home. MDSC adoptively transferred into either normal or tumor-bearing mice homed mainly to the liver and spleen (Fig. 1A). Since MDSC had not been previously described to home to the liver, we investigated whether MDSC normally accumulate in the livers of tumor-bearing mice. Injecting a fluorescently labeled GR-1 antibody detected by a bioimager, we found that greater levels of antibody were detected in the livers of tumor-bearers, relative to those of normals, as well as in the bone and spleen as previously described sites of MDSC accumulation (Fig. 1B). Histology of the livers from tumor bearers show nests of myeloid cells peri-vascularly as well as throughout the liver parenchyma of tumor-bearers (Fig. 1C and Fig.2)), and flow cytometry confirmed the presence of high levels of CD11b+GR-1+ cells (Fig. 3).
There have been previous reports of hematopoiesis in livers of tumor-bearers, but the cells were either not specifically identified or labeled as granulocytes and more importantly were attributed to liver metastatic disease by the 4T1 breast tumor model (36, 37). More recently, Li et al have identified MDSC accumulation in the livers of an orthotopic liver cancer model, where tumor cells are injected directly into the liver (38). While there have been several reports like these of MDSC within tumors, and being recruited to tumor cells, our data show that MDSC accumulate in the livers of both tumor models with metastatic disease to the liver (4T1), and in the absence of liver metastatic disease (DA-3, B16, LLC). We also showed that liver MDSC are functionally suppressive of T cell proliferation, similar to those from the spleen (Fig. 3C). Furthermore, GM-CSF which has been shown to be secreted by various human and mouse cancers was able to expand MDSC in the livers in the absence of a growing tumor (Fig. 4A). Although we showed in Figure 1 that MDSC home to the liver after adoptive transfer, based on the nesting pattern on liver histology it raised the possibility that MDSC could also be arising from the liver. Other groups have shown that splenic hematopoiesis, by the presence of myeloid progenitors and their clonogenic activity, was highly up-regulated in tumor bearers (19). We confirmed that the bone marrow and spleen had high levels of hematopoietic activity, but also found that the liver likewise has higher activity in tumor-bearing mice (Fig. 4B). Thus all these sites contribute to the expansion of MDSC in these organs.
We and others have shown that macrophages in tumor-bearers are altered systemically (39-41), and since the liver is a site for macrophages known as Kupffer cells, we analyzed whether their phenotype was affected in tumor bearers. PD-L1 is a negative costiumulatory molecule expressed in the liver by liver sinusoidal endothelial cells (42), Kupffer cells (42), and hepatocytes (43), and all have been shown to be capable of down-regulating T cell responses and activation via this negative regulator. Furthermore, blocking this negative regulatory pathway has been shown to enhance immunity in certain pathological states, such as viral clearance (42). Analyzing the levels of PD-L1 on Kupffer cells, we found a twofold higher expression on cells from tumor-bearing mice (Fig. 5). This up-regulation was specific to these liver macrophages, as splenic macrophages did not show a difference in PD-L1 expression. Furthermore, adoptive transfer of tumor bearers’ splenocytes or purified MDSC, from tumor bearers’ spleens or pooled normal spleens, into normal mice resulted in an up-regulation of PD-L1 on Kupffer cells. Thus MDSC within the liver interact with Kupffer cells to up-regulate their expression of PD-L1. It is also important to note that although in some reports PD-L1 expression was found on MDSC (44), in the tumor models we tested MDSC were consistently negative for PD-L1 while Kupffer cells express relatively high levels. This may be due to a difference in tumor-derived factors between the tumor types, which may regulate expression of PD-L1 on MDSC. The data presented also confirms previous reports that PD-L1 expression by Kupffer cells suppresses IFN-γ production by activated T cells, and thus potentially limits effective T cell dependent immune responses (Fig. 6).
This study reveals that systemic levels of MDSC are up-regulated in livers of tumor-bearing mice, and that these cells can home to this organ in addition to other previously reported organ sites. The observed nests of MDSC in the livers of tumor bearers, and the increased hematopoietic clonogenic activity, also suggest the possibility that they may develop in the liver in addition to just accumulating there. Furthermore, MDSC in the liver interact with Kupffer cells and augment their expression of PD-L1, a negative costimulatory molecule. MDSC in the liver likely also modulate other cells as this organ is known to harbor various immune-related cells, as was recently shown for MDSC interactions with liver NK cells (38). These findings open up new avenues to understanding how MDSC regulate immunity, as their effects on other cell populations can be organ-specific, and our understanding of their biology is applicable to various areas where they have been described in addition to cancer, such as inflammation (7), transplantation (11), and infection (10).
We are particularly grateful to Drs. Carolyn Cray and Norman H. Altman from Comparative Pathology for their assistance and advice with tissue histology, Joy Prewitt from the Oncogenomics Core facility for help with the IVIS bioimager, and Jim Phillips from the Flow Cytometry Core facility. We also thank Taylor Schreiber for help with confocal imaging.
Supported by grants R01CA25583, and F31GM079805 from the National Institutes of Health, National Cancer Center pre-doctoral fellowship, and BD Biosciences Immunology Research Grant.