Soluble NKG2D ligand promotes MDSC expansion and skews macrophage to the alternatively activated phenotype
© Xiao et al.; licensee BioMed Central. 2015
Received: 8 December 2014
Accepted: 14 January 2015
Published: 20 February 2015
Expression of surface NKG2D ligand MIC on tumor cells is deemed to stimulate NK and co-stimulate CD8 T cell anti-tumor immunity. Human cancer cells however frequently adopt a proteinase-mediated shedding strategy to generate soluble MIC (sMIC) to circumvent host immunity. High levels of sMIC have been shown to correlate with advanced disease stages in cancer patients. The underlying mechanism is currently understood as systemic downregulation of NKG2D expression on CD8 T and NK cells and perturbing NK cell periphery maintenance. Herein we report a novel mechanism by which sMIC poses immune suppressive effect on host immunity and tumor microenvironment. We demonstrate that sMIC facilitates expansion of myeloid-derived suppressor cells (MDSCs) and skews macrophages to the more immune suppressive alternative phenotype through activation of STAT3. These findings further endorse that sMIC is an important therapeutic target for cancer immunotherapy.
Expression of surface NKG2D ligands on tumor cells is well proven to provoke tumor rejection through activation of NK and CD8 T cells in experimental animal models [1-3]. Human cancers however broadly evade this mechanism by adopting a protease-dependent shedding mechanism to generate soluble NKG2D ligands [4-6]. Among the many identified human NKG2D ligands , the MHC I chain-related molecule (MIC) is the best described and characterized ligands in human cancer. Elevated shedding of cell surface MIC has been associated with advanced disease stages and metastasis in many types of epithelial cancers [4,7-10]. It is well accepted that tumor-derived soluble MIC (sMIC) is a negative immune regulator in cancer patients, although the underlying mechanisms are not fully understood.
Multiple mechanisms have been uncovered by which soluble sMIC insults the immune system. Tumor-derived sMIC has been shown broadly downregulating NKG2D expression on NK, CD8 T, NKT, and γδ T cells and thus impair their effector function [4,10-12]. Very recently, we further show that tumor-derived sMIC perturbs NK cell periphery homoeostatic maintenance through disrupting the ability of NK cell to self-renew . In this study, we report a novel mechanism by which tumor-derived sMIC negatively regulates host immunity and the immune reactive tumor microenvironment. With complementary in vivo and in vitro assays, we demonstrate that sMIC facilitates expansion of myeloid-derived suppressor cells (MDSCs) and skews macrophages to the alternative immune suppressive phenotype through activation of STAT3.
Results and discussion
sMICB increases frequency of MDSC and arginase I+ cells in bi-transgenic TRAMP/MIC mice
MIC is not expressed in rodents, which limits the potential to study the global impact of tumor-derived sMIC on host anti-tumor immunity in vivo. To overcome this limitation and to take the advantage that human MICB can serve as a mouse NKG2D ligand and activate mouse NKG2D [2,13-15], we have recently generated a bi-transgenic TRAMP/MICB spontaneous prostate tumor model in which the native form of human MICB was specifically expressed in the autochthonous transgenic adenocarcinoma mouse prostate (TRAMP) under the prostate-specific promoter . The TRAMP/MICB model highly resembles human cancer patients in the kinetics of oncogenesis and tumor immunity in that MIC expression is concurrent with oncogenic events and MIC shedding correlates with tumor progression .
Intraperitoneal injection of sMIC promotes local accumulation of MDSCs and macrophages with alternative phenotype
MDSCs are a collective of heterogeneous population of immature myeloid cells (IMC) that are endowed with a robust immunosuppressive activity through secretion of immune suppressive factors, such as inducible nitric oxide synthase (iNOS), arginase, and indoleamine 2,3-dioxygnases (IDOs) . The heterogeneous MDSC population are generally defined by surface marker CD11b+GR-1+ and considered to be immune suppressive in tumor host. MDSCs can be expanded during cancer development due to blockage of normal myeloid cell differentiation and maturation by tumor or tumor stroma-derived intrinsic factors .
sMICB promotes induction of MDSC in vitro through NKG2D and activation of STAT3
We next addressed whether sMIC can induce MDSC accumulation in vitro in the absence of tumor cells. We cultured bone marrow cells with various concentrations of purified sMICB in the presence of GM-CSF and analyzed the cells at day 3 of culture. As representatively shown in Figure 3b, sMIC elicited a dose-dependent effect on the induction of Gr-1+CD11b+ cells. NKG2D, the only known cell surface receptor for sMIC, was detected on the surface MDSCs, with a trend of increased expression after exposure to GM-CSF (Figure 3c). We thus further asked whether NKG2D is necessary for the effect of sMIC in the current experimental setting. In the presence of the NKG2D blocking antibody CX5, sMIC failed to augment MDSC expansion (Figure 3d). This observation was substantiated by experiments demonstrating that sMIC has no effect on bone marrow cells from NKG2D−/− mice (Additional file 4: Figure S4a). Together, these data confirmed a direct effect of sMIC on the accumulation of myeloid cells with MDSC phenotypes (Gr-1+CD11b+).
We sought to further understand the molecular pathways under which sMIC induces MDSC accumulation. MDSC expansion can be triggered by multiple factors that include cyclooxygenase-2(COX2), prostaglandins , stem cell factor (SCF) , macrophage colony-stimulating factor (M-CSF), IL-6 , GM-CSF , and vascular endothelial growth factors . Signaling pathways triggered by most of these factors converge to the activation of the signal transducer and activator of transcription 3 (STAT3) , which is the main transcriptional factor regulating MDSC expansion [27,28]. MDSCs from tumor bearing mice showed markedly increased levels of phosphorylated STAT3 (pSTAT3) compared with IMCs from naïve mice . As shown in Figure 3d, addition of the STAT3 inhibitor AG490 not only mitigated the effect of sMICB on MDSC accumulation but also nearly obliterated MDSC expansion (Figure 3d). Concurrently, the intracellular levels of pSTAT3 was decreased to base-level with anti-NKG2D blocking antibody CX5 and abolished with pSTAT3 inhibitor AG490 (Figure 3e). Given that GM-CSF induce potent STAT3 activation and that STAT3 is a key transcriptional factor in regulating MDSC expansion [19,20], the much more profound effect of AG490 than anti-NKG2D antibody on MDSC expansion and STAT3 is anticipated. No significant change in the levels of STAT3 phosphorylation was induced by sMICB in the same experiment settings with bone marrow cells from NKG2D−/− mice  (Additional file 4: Figure S4b). Collectively, these data demonstrate that sMIC induces MDSC expansion through activation of STAT3 pathways.
sMIC activation of STAT3 skews macrophage differentiation into to the alternative phenotype
To this end, we demonstrate that tumor-derived soluble NKG2D ligand sMICB facilitates the expansion of MDSC and skews macrophage differentiation into an alternatively active phenotype through NKG2D and activation of the STAT3 pathway. Classically, activation of a PI3K/Grb2-Vav pathway is the canonical outcome of NKG2D signaling via coupling to the adaptor molecule DAP10 in both mouse and human cells [31,32], although NKG2D in mouse, ambiguously in human, has been shown to promiscuously and selectively associate with DAP10 or DAP12 adaptor molecule, depending on the availability of the adaptor partners, cell types, and cell activation status [33-36]. Whether NKG2D signaling in MDSC or activated macrophage primarily activates the PI3K pathway with subsequent activation of STAT3 pathways or directly activates the STAT3 pathway is an interesting question to be further investigated. It has been shown that PI3K signaling can bisect at phosphatidylinositol 3,4,5-trisphosphate (PIP3) to activate the non-receptor bone-marrow tyrosine kinase BTK and the downstream STAT3 signaling during cellular oncogenic transformation . Provided with this knowledge, it is reasonable to speculate that activation of STAT3 pathways via NKG2D signaling in the bone marrow-originated MDSCs and macrophages lies downstream of the canonical PI3K signaling. It would be an interesting future investigation to test this speculation with specific PI3K or BTK inhibitors.
Tumor-derived soluble NKG2D ligands, namely sMIC, is known to negatively impact host immune response by downregulating NKG2D expression on effector NK and CD8 T cells and most recently shown by perturbing NK cell peripheral maintenance. Here we demonstrate that sMIC can also promote the expansion of MDSC and skew macrophage into a more immune suppressive phenotype in a given microenvironment. We demonstrated that sMIC can activate STAT3 pathway in cells of myeloid lineage to foster their immune suppressive potential. We have demonstrated a novel mechanism by which tumor-derived sMIC may temper the immune reactive tumor microenvironment. Our findings further emphasize the significance of targeting sMIC for cancer immune therapy.
Reagents, constructs, and cell lines
STAT3 inhibitor AG490 was purchased from Invivogen (San Diego, CA, USA). cDNA containing the α1-3 ectodomain of MICB fused with HIS6-Tag at the C-terminus was synthesized by GENEWIZ Inc. (South Plainfield, NJ, USA) and subcloned into the retroviral expression vector pBMN-GFP as described previously . The sMICB expression plasmid pBMN-GFP-sMICB or control vector pBMN-GFP was transfected into 293 T cells. After 2 weeks of selection with puromycin (5 μg/ml), GFP-positive cells were isolated by flow cytometry sorting. Serum-free culture supernatant of sorted GFP+ 293 T cells was collected and subjected to Ni-sepharose Fast Flow column from (GE Healthcare, Pewaukee, WI, USA) and eluted with imidazole gradient increase method according to manufacturer’s instruction. The elution was loaded onto the Hydrophobic Interaction Chromatography (HIC) column (GE Healthcare) for size fractionation. The final collected fraction was buffer exchange with the PBS (pH 7.2). The purity of sMICB after buffer exchange was assayed by SDS-PAGE gel and Western-blotting with anti-MIC antibody (Additional file 6: Figure S6). Specific binding of purified recombinant sMICB to mouse NKG2D was confirmed by flow cytometry analyses (Additional file 6: Figure S6). Generation of sMICB-expressing TRAMP-C2 cells was described previously . Tumor cell lines TRAMP-C2, derivatives, and mouse fibroblast L929 cell line were maintained in Dulbecco modified Eagle medium (DMEM) plus 10% fetal bovine serum and antibiotics. Conditioned media of L929 cells was collected, centrifuged, and filtered through a 0.22-μm filter before being applied to bone marrow culture.
Mice and in vitro bone marrow differentiation
All animal procedures were approved by the MUSC Institutional Animal Care and Use Committee. 7- to 10-week-old male C57BL/6 and BALB/c mice were purchased from The Jackson Laboratory (Bar Harbor, ME, USA). NKG2D-deficient mice (gift of Dr. David Raulet, University of California at Berkeley) were bred in house. All mice were housed in Specific Pathogen Free facility. Single cell suspension of bone marrow (BM) cells were plated at the density of 3 × 106 cells/ml in DMEM supplemented with 10% fetal bovine serum, 50 mM 2-mercaptoethanol, 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer (Life technologies, Grand Island, NY, USA), 1 mM sodium pyruvate, 100 U/ml penicillin, 100 mg/ml streptomycin, and amino acids (1.5 mM L-glutamine, L-arginine, and L-asparagine), with or without 1 ng/ml rmGM-CSF (Biolegend, San Diego, CA, USA). sMICB protein of designated concentration was added where indicated. In some experiments, bone marrow cells were cultured with conditioned media derived from L929 cell lines.
Peritoneal cell isolation
Mice were injected via intraperitoneal (i.p) routine with sMICB or control PBS flow through from the culture supernatant of 293 F cells that were transduced with control vector expressing HIS6-Tag alone. At indicated time points, animals were euthanized and the peritoneal exudate cells (PECs) were harvested by lavage with 10 ml of ice-cold PBS.
Cells were stained using combination of fluorochrome-conjugated anti-mouse CD11b and Gr-1 antibody to define the general population of MDSC and fluorochrome-conjugated anti-mouse F4/80 in combination with CD206 and CD11c and intracellular staining of arginase I to define the phenotype of macrophage. All fluorescence-conjugated antibodies were from eBioscience except for rabbit anti-pSTAT3 (Cell signaling, Danvers, MA, USA). Appropriately conjugated species and isotype-matched IgG was used as staining controls. Multi-colored flow cytometry data were collected on a BD LSRII flow cytometer and analyzed with FlowJo software (Tree Star).
All results are expressed as the mean ± SEM. Differences between the mean of groups were analyzed using student’s t-test. P < 0.05 was considered as significant.
This work was supported by NIH grant 1R01CA149405 and Prostate Cancer Foundation Challenge Award and the Mazzone Program Funded by The United States District Court for the District of Massachusetts from 2004 Lupron Settlement Fund. We thank Hollings Cancer Center Flow Cytometry Core Facility. We thank John Jarzen for critically reading the manuscript.
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