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1.  MDSC as a Mechanism of Tumor Escape from Sunitinib Mediated Anti-Angiogenic Therapy 
International immunopharmacology  2011;11(7):853-858.
Sunitinib is a receptor tyrosine kinase inhibitor (TKI) that is front-line therapy for metastatic renal cell carcinoma (mRCC). Its antitumor activity is related to its ability to block tumor cell and tumor vasculature cell signaling via several TKI receptors (i.e. vascular endothelial growth factor receptors VEGFRs, platelet-derived growth factors (PDGFs), stem cell factors). Sunitinib also targets myeloid derived suppressor cells (MDSCs) significantly reducing their accumulation in the peripheral blood and reversing T cell (IFNγ) suppression in both mRCC patients and in murine tumor models. This reduction in immune suppression provides a rationale for combining sunitinib with immunotherapy for the treatment of certain tumor types. Despite these encouraging findings, however, we have observed that sunitinib has variable impact at reducing MDSCs and restoring T cell function within the tumor microenvironment. Given the immunosuppressive and proangiogenic activities of MDSC, it seems plausible that their persistence may contribute to the resistance that develops in sunitinib-treated patients. While sunitinib reduced tumor infiltrating MDSCs in Renca and CT26-bearing mice, coinciding with strong to modest decreases in tumor size respectively, it was ineffective at ‘reducing MDSCs (<35% reduction in Gr1+CD11b+) or tumor burden in 4T1-bearing mice. Persistence of intratumor MDSCs was paralleled by depressed intratumor T cell IFNγ response and increased GM-CSF expression. Additionally, in vitro and in vivo experiments showed that GM-CSF prolongs survival of MDSCs, thus protecting them from the effects of sunitinib via a pSTAT5-dependent pathway. Although preliminary, there is evidence of intratumor MDSC resistance in some mRCC patients following sunitinib treatment. Intratumor MDSC persistence and T cell IFNγ response post nephrectomy in patients receiving sunitinib in a neoadjuvant setting is being compared to RCC patients undergoing nephrectomy without prior sunitinib treatment. Tumors from untreated patients showed suppressed T cell IFNγ response along with substantial expression of MDSCs (5% of total digested cells). Thus far, tumors from 5/8 neoadjuvant patients showed persistence of intratumor MDSCs and low T cell IFNγ production post sunitinib treatment, findings that parallel results from untreated tumors. In the remaining 3 neoadjuvant patients, intratumor MDSCs were detected at low levels which coincided with a T cell IFNγ response similar to that observed with normal donor peripheral T cells. GM-CSF’s role in promoting MDSC survival in patient tumors is supported by the observation that GM-CSF is produced in short-term RCC cultures at levels capable of protecting MDSCs from sunitinib-induced cell death. Additionally, persistence of MDSC also may be associated with increased expression of proangiogenic proteins, such as MMP9, MMP8, and IL-8 produced by tumor stromal cells or infiltrating MDSCs. Indeed our findings suggest that the most dominate MDSC subset in RCC patients is the neutrophilic population that produces proangiogenic proteins. We propose that the development of sunitinib resistance is partly mediated by the survival of MDSCs intratumorally, thereby providing sustained immune suppression and angiogenesis.
doi:10.1016/j.intimp.2011.01.030
PMCID: PMC3109226  PMID: 21315783
2.  Tumor-Derived G-CSF Facilitates Neoplastic Growth through a Granulocytic Myeloid-Derived Suppressor Cell-Dependent Mechanism 
PLoS ONE  2011;6(11):e27690.
Myeloid-derived suppressor cells (MDSC) are induced under diverse pathologic conditions, including neoplasia, and suppress innate and adaptive immunity. While the mechanisms by which MDSC mediate immunosuppression are well-characterized, details on how they develop remain less understood. This is complicated further by the fact that MDSC comprise multiple myeloid cell types, namely monocytes and granulocytes, reflecting diverse stages of differentiation and the proportion of these subpopulations vary among different neoplastic models. Thus, it is thought that the type and quantities of inflammatory mediators generated during neoplasia dictate the composition of the resultant MDSC response. Although much interest has been devoted to monocytic MDSC biology, a fundamental gap remains in our understanding of the derivation of granulocytic MDSC. In settings of heightened granulocytic MDSC responses, we hypothesized that inappropriate production of G-CSF is a key initiator of granulocytic MDSC accumulation. We observed abundant amounts of G-CSF in vivo, which correlated with robust granulocytic MDSC responses in multiple tumor models. Using G-CSF loss- and gain-of-function approaches, we demonstrated for the first time that: 1) abrogating G-CSF production significantly diminished granulocytic MDSC accumulation and tumor growth; 2) ectopically over-expressing G-CSF in G-CSF-negative tumors significantly augmented granulocytic MDSC accumulation and tumor growth; and 3) treatment of naïve healthy mice with recombinant G-CSF protein elicited granulocytic-like MDSC remarkably similar to those induced under tumor-bearing conditions. Collectively, we demonstrated that tumor-derived G-CSF enhances tumor growth through granulocytic MDSC-dependent mechanisms. These findings provide us with novel insights into MDSC subset development and potentially new biomarkers or targets for cancer therapy.
doi:10.1371/journal.pone.0027690
PMCID: PMC3218014  PMID: 22110722
3.  Myeloid-derived suppressor cells: general characteristics and relevance to clinical management of pancreatic cancer 
Current cancer drug targets  2011;11(6):734-751.
Recent studies describe a heterogeneous population of cells of the myeloid lineage, termed myeloid derived suppressor cells (MDSC), which are observed with increased prevalence in the peripheral blood and tumor microenvironment of cancer patients, including pancreatic cancer. Accumulation of MDSC in the peripheral circulation has been related to extent of disease, and correlates with stage. MDSC have primarily been implicated in promoting tumor growth by suppressing antitumor immunity. There is also compelling evidence MDSC are also involved in angiogenesis and metastatic spread.
Two main subsets of MDSC have been identified in cancer patients: a monocytic subset, characterized by expression of CD14, and a granulocytic subset characterized by expression of CD15. Both subsets of MDSC actively suppress host immunity through a variety of mechanisms including production of reactive oxygen species and arginase. Just as in humans, accumulation of monocytic and granulocytic MDSC has been noted in the bone marrow, spleen, peripheral circulation, and tumors of tumor bearing mice.
Successful targeting of MDSC in mice is associated with improved immune responses, delayed tumor growth, improved survival, and increased efficacy of vaccine therapy. By further elucidating mechanisms of MDSC recruitment and maintenance in the tumor environment, strategies could be developed to reverse immune tolerance to tumor. We discuss here what is currently known about MDSC as well as some potential strategies targeting MDSC in the context of our work on pancreatic cancer and recent literature. Due to the number of new reports on MDSC, the most pertinent ones have been selected.
PMCID: PMC3670669  PMID: 21599634
myeloid-derived suppressor cells; pancreatic cancer; immune suppression; bone marrow; metastasis
4.  Myeloid-derived suppressor cell development is regulated by a STAT/IRF-8 axis 
The Journal of Clinical Investigation  2013;123(10):4464-4478.
Myeloid-derived suppressor cells (MDSCs) comprise immature myeloid populations produced in diverse pathologies, including neoplasia. Because MDSCs can impair antitumor immunity, these cells have emerged as a significant barrier to cancer therapy. Although much research has focused on how MDSCs promote tumor progression, it remains unclear how MDSCs develop and why the MDSC response is heavily granulocytic. Given that MDSCs are a manifestation of aberrant myelopoiesis, we hypothesized that MDSCs arise from perturbations in the regulation of interferon regulatory factor–8 (IRF-8), an integral transcriptional component of myeloid differentiation and lineage commitment. Overall, we demonstrated that (a) Irf8-deficient mice generated myeloid populations highly homologous to tumor-induced MDSCs with respect to phenotype, function, and gene expression profiles; (b) IRF-8 overexpression in mice attenuated MDSC accumulation and enhanced immunotherapeutic efficacy; (c) the MDSC-inducing factors G-CSF and GM-CSF facilitated IRF-8 downregulation via STAT3- and STAT5-dependent pathways; and (d) IRF-8 levels in MDSCs of breast cancer patients declined with increasing MDSC frequency, implicating IRF-8 as a negative regulator in human MDSC biology. Together, our results reveal a previously unrecognized role for IRF-8 expression in MDSC subset development, which may provide new avenues to target MDSCs in neoplasia.
doi:10.1172/JCI68189
PMCID: PMC3784535  PMID: 24091328
5.  Pancreatic adenocarcinoma induces bone marrow mobilization of myeloid derived suppressor cells which promote primary tumor growth 
Purpose
Myeloid derived suppressor cells (MDSC) are a heterogeneous population of immunosuppressive cells that are upregulated in cancer. Little is known about the prevalence and importance of MDSC in pancreas adenocarcinoma (PA).
Experimental Design
Peripheral blood, bone marrow, and tumor samples were collected from pancreatic cancer patients, analyzed for MDSC (CD15+CD11b+) by flow cytometry, and compared to cancer free controls. The suppressive capacity of MDSC (CD11b+Gr-1+) and the effectiveness of MDSC depletion were assessed in C57BL/6 mice inoculated with Pan02, a murine PA, and treated with placebo or zoledronic acid, a potent aminobisphosphonate previously shown to target MDSC. The tumor microenvironment was analyzed for MDSC (Gr1+CD11b+), effector T cells, and tumor cytokine levels.
Results
Patients with PA demonstrated increased frequency of MDSC in the bone marrow and peripheral circulation which correlated with disease stage. Normal pancreas tissue showed no MDSC infiltrate, while human tumors avidly recruited MDSC. Murine tumors similarly recruited MDSC that suppressed CD8+ T cells in vitro and accelerated tumor growth in vivo. Treatment with zoledronic acid impaired intratumoral MDSC accumulation resulting in delayed tumor growth rate, prolonged median survival, and increased recruitment of T cells to the tumor. This was associated with a more robust type 1 response with increased levels of IFN-γ and decreased levels of IL-10.
Conclusions
MDSC are important mediators of tumor-induced immunosuppression in pancreatic cancer. Inhibiting MDSC accumulation with zoledronic acid improves the host anti-tumor response in animal studies suggesting that efforts to block MDSC may represent a novel treatment strategy for pancreatic cancer.
doi:10.1007/s00262-011-1178-0
PMCID: PMC3697836  PMID: 22215137
myeloid derived suppressor cells; pancreatic cancer; zoledronic acid
6.  ER stress regulates myeloid-derived suppressor cell fate through TRAIL-R–mediated apoptosis 
The Journal of Clinical Investigation  2014;124(6):2626-2639.
Myeloid-derived suppressor cells (MDSCs) dampen the immune response thorough inhibition of T cell activation and proliferation and often are expanded in pathological conditions. Here, we studied the fate of MDSCs in cancer. Unexpectedly, MDSCs had lower viability and a shorter half-life in tumor-bearing mice compared with neutrophils and monocytes. The reduction of MDSC viability was due to increased apoptosis, which was mediated by increased expression of TNF-related apoptosis–induced ligand receptors (TRAIL-Rs) in these cells. Targeting TRAIL-Rs in naive mice did not affect myeloid cell populations, but it dramatically reduced the presence of MDSCs and improved immune responses in tumor-bearing mice. Treatment of myeloid cells with proinflammatory cytokines did not affect TRAIL-R expression; however, induction of ER stress in myeloid cells recapitulated changes in TRAIL-R expression observed in tumor-bearing hosts. The ER stress response was detected in MDSCs isolated from cancer patients and tumor-bearing mice, but not in control neutrophils or monocytes, and blockade of ER stress abrogated tumor-associated changes in TRAIL-Rs. Together, these data indicate that MDSC pathophysiology is linked to ER stress, which shortens the lifespan of these cells in the periphery and promotes expansion in BM. Furthermore, TRAIL-Rs can be considered as potential targets for selectively inhibiting MDSCs.
doi:10.1172/JCI74056
PMCID: PMC4038578  PMID: 24789911
7.  Down-regulation of PLCγ2–β-catenin pathway promotes activation and expansion of myeloid-derived suppressor cells in cancer 
The Journal of Experimental Medicine  2013;210(11):2257-2271.
Down-regulation of PLCγ2 and β-catenin signaling in tumor associated myeloid cells allows for their expansion and tumor growth in mice and humans.
Myeloid-derived suppressor cells (MDSCs) favor tumor promotion, mainly by suppressing antitumor T cell responses in many cancers. Although the mechanism of T cell inhibition is established, the pathways leading to MDSC accumulation in bone marrow and secondary lymphoid organs of tumor-bearing hosts remain unclear. We demonstrate that down-regulation of PLCγ2 signaling in MDSCs is responsible for their aberrant expansion during tumor progression. PLCγ2−/− MDSCs show stronger immune-suppressive activity against CD8+ T cells than WT MDSCs and potently promote tumor growth when adoptively transferred into WT mice. Mechanistically, PLCγ2−/− MDSCs display reduced β-catenin levels, and restoration of β-catenin expression decreases their expansion and tumor growth. Consistent with a negative role for β-catenin in MDSCs, its deletion in the myeloid population leads to MDSC accumulation and supports tumor progression, whereas expression of β-catenin constitutively active reduces MDSC numbers and protects from tumor growth. Further emphasizing the clinical relevance of these findings, MDSCs isolated from pancreatic cancer patients show reduced p-PLCγ2 and β-catenin levels compared with healthy controls, similar to tumor-bearing mice. Thus, for the first time, we demonstrate that down-regulation of PLCγ2–β-catenin pathway occurs in mice and humans and leads to MDSC-mediated tumor expansion, raising concerns about the efficacy of systemic β-catenin blockade as anti-cancer therapy.
doi:10.1084/jem.20130281
PMCID: PMC3804931  PMID: 24127488
8.  Antigen-specific CD4+ T cells regulate function of myeloid-derived suppressor cells in cancer via retrograde MHC class II signaling 
Cancer research  2012;72(4):928-938.
Myeloid-derived suppressor cells (MDSC) play a major role in cancer-related immune suppression, yet the nature of this suppression remains controversial. In this study, we evaluated the ability of MDSC to elicit CD4+ T cell tolerance in different mouse tumor models. In contrast to CD8+ T-cell tolerance, which could be induced by MDSC in all the tumor models tested, CD4+ T-cell tolerance could be elicited in only one of the models (MC38) where a substantial level of MHC class II was expressed on MDSC, compared to control myeloid cells. Mechanistic investigations revealed that MDSC deficient in MHC class II could induce tolerance to CD8+ T cells but not to CD4+ T cells. Unexpectedly, antigen-specific CD4+ T cells (but not CD8+ T cells) could dramatically enhance the immune suppressive activity of MDSC by converting them into powerful non-specific suppressor cells. This striking effect was mediated by direct cell-cell contact through cross-linking of MHC class II on MDSC. We also implicated an Ets-1 transcription factor-regulated increase in expression of Cox-2 and prostaglandin E2 in MDSCs in mediating this effect. Together, our findings suggest that activated CD4+ T cells that are antigen-specific may enhance the immune suppressive activity of MDSC, a mechanism that might serve normally as a negative feedback loop to control immune responses that becomes dysregulated in cancer.
doi:10.1158/0008-5472.CAN-11-2863
PMCID: PMC4062074  PMID: 22237629
9.  Functional characterization of human Cd33+ And Cd11b+ myeloid-derived suppressor cell subsets induced from peripheral blood mononuclear cells co-cultured with a diverse set of human tumor cell lines 
Background
Tumor immune tolerance can derive from the recruitment of suppressor cell populations, including myeloid-derived suppressor cells (MDSC). In cancer patients, MDSC accumulation correlates with increased tumor burden, but the mechanisms of MDSC induction remain poorly understood.
Methods
This study examined the ability of human tumor cell lines to induce MDSC from healthy donor PBMC using in vitro co-culture methods. These human MDSC were then characterized for morphology, phenotype, gene expression, and function.
Results
Of over 100 tumor cell lines examined, 45 generated canonical CD33+HLA-DRlowLineage- MDSC, with high frequency of induction by cervical, ovarian, colorectal, renal cell, and head and neck carcinoma cell lines. CD33+ MDSC could be induced by cancer cell lines from all tumor types with the notable exception of those derived from breast cancer (0/9, regardless of hormone and HER2 status). Upon further examination, these and others with infrequent CD33+ MDSC generation were found to induce a second subset characterized as CD11b+CD33lowHLA-DRlowLineage-. Gene and protein expression, antibody neutralization, and cytokine-induction studies determined that the induction of CD33+ MDSC depended upon over-expression of IL-1β, IL-6, TNFα, VEGF, and GM-CSF, while CD11b+ MDSC induction correlated with over-expression of FLT3L and TGFβ. Morphologically, both CD33+ and CD11b+ MDSC subsets appeared as immature myeloid cells and had significantly up-regulated expression of iNOS, NADPH oxidase, and arginase-1 genes. Furthermore, increased expression of transcription factors HIF1α, STAT3, and C/EBPβ distinguished MDSC from normal counterparts.
Conclusions
These studies demonstrate the universal nature of MDSC induction by human solid tumors and characterize two distinct MDSC subsets: CD33+HLA-DRlowHIF1α+/STAT3+ and CD11b+HLA-DRlowC/EBPβ+, which should enable the development of novel diagnostic and therapeutic reagents for cancer immunotherapy.
doi:10.1186/1479-5876-9-90
PMCID: PMC3128058  PMID: 21658270
myeloid-derived suppressor cells; tumor immune tolerance; human tumor cell lines; immunomodulation; cytokines; hypoxia-inducible factor 1 alpha; CAAAT-enhancer binding protein; signal transducer and activator of transcription; inflammation
10.  Subsets of Myeloid-Derived Suppressor Cells in Tumor Bearing Mice1 
Myeloid-derived suppressor cells (MDSC) are a heterogeneous group of cells that play a critical role in tumor associated immune suppression. In an attempt to identify a specific subset of MDSC primarily responsible for immunosuppressive features of these cells, 10 different tumor models were investigated. All models showed variable but significant increase in the population of MDSC. Variability of MDSC expansion in vivo matched closely the effect of tumor-cell condition media (TCCM) in vitro. MDSC consists of two major subsets of Ly6G+Ly6Clow granulocytic and Ly6G-Ly6Chigh monocytic cells. Granulocytic MDSC have increased level of reactive oxygen species (ROS) and undetectable level of nitric oxide (NO) whereas monocytic MDSC had increased level of NO but undetectable levels of ROS. However, their suppressive activity per cell basis was comparable. Almost all tumor models demonstrated a preferential expansion of granulocytic subset of MDSC. We performed a phenotypical and functional analysis of several surface molecules previously suggested to be involved in MDSC mediated suppression of T cells: CD115, CD124, CD80, PD-L1, and PD-L2. Although substantial proportion of MDSC expressed those molecules no differences in the level of their expression or the proportion, positive cells were found between MDSC and cells from tumor-free mice that lack immune suppressive activity. The level of MDSC mediated T-cell suppression did not depend on the expression of these molecules. This data indicates that suppressive features of MDSC is caused not by expansion of a specific subset but more likely represents a functional state of these cells.
PMCID: PMC2575748  PMID: 18832739
rodent; myeloid-derived suppressor cells; tumor immunity
11.  Immunosuppressive activity enhances central carbon metabolism and bioenergetics in myeloid-derived suppressor cells in vitro models 
BMC Cell Biology  2012;13:18.
Background
The tumor microenvironment contains a vast array of pro- and anti-inflammatory cytokines that alter myelopoiesis and lead to the maturation of immunosuppressive cells known as myeloid-derived suppressor cells (MDSCs). Incubating bone marrow (BM) precursors with a combination of granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-6 (IL-6) generated a tumor-infiltrating MDSC-like population that impaired anti-tumor specific T-cell functions. This in vitro experimental approach was used to simulate MDSC maturation, and the cellular metabolic response was then monitored. A complementary experimental model that inhibited L-arginine (L-Arg) metabolizing enzymes in MSC-1 cells, an immortalized cell line derived from primary MDSCs, was used to study the metabolic events related to immunosuppression.
Results
Exposure of BM cells to GM-CSF and IL-6 activated, within 24 h, L-Arg metabolizing enzymes which are responsible for the MDSCs immunosuppressive potential. This was accompanied by an increased uptake of L-glutamine (L-Gln) and glucose, the latter being metabolized by anaerobic glycolysis. The up-regulation of nutrient uptake lead to the accumulation of TCA cycle intermediates and lactate as well as the endogenous synthesis of L-Arg and the production of energy-rich nucleotides. Moreover, inhibition of L-Arg metabolism in MSC-1 cells down-regulated central carbon metabolism activity, including glycolysis, glutaminolysis and TCA cycle activity, and led to a deterioration of cell bioenergetic status. The simultaneous increase of cell specific concentrations of ATP and a decrease in ATP-to-ADP ratio in BM-derived MDSCs suggested cells were metabolically active during maturation. Moreover, AMP-activated protein kinase (AMPK) was activated during MDSC maturation in GM-CSF and IL-6–treated cultures, as revealed by the continuous increase of AMP-to-ATP ratios and the phosphorylation of AMPK. Likewise, AMPK activity was decreased in MSC-1 cells when L-Arg metabolizing enzymes were inhibited. Finally, inhibition of AMPK activity by the specific inhibitor Compound C (Comp-C) resulted in the inhibition of L-Arg metabolizing enzyme activity and abolished MDSCs immunosuppressive activity.
Conclusions
We anticipate that the inhibition of AMPK and the control of metabolic fluxes may be considered as a novel therapeutic target for the recovery of the immunosurveillance process in cancer-bearing hosts.
doi:10.1186/1471-2121-13-18
PMCID: PMC3433355  PMID: 22762146
Myeloid-derived suppressor cells; GM-CSF; IL-6; MSC-1 cells; Central carbon metabolism; Bioenergetics
12.  The Liver is a Site for Tumor Induced Myeloid-Derived Suppressor Cell Accumulation and Immunosuppression 
Cancer research  2009;69(13):5514-5521.
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.
doi:10.1158/0008-5472.CAN-08-4625
PMCID: PMC2706931  PMID: 19549903
13.  Identification of myeloid-derived suppressor cells in the synovial fluid of patients with rheumatoid arthritis: a pilot study 
Background
Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of innate immune cells with a granulocyte-like or monocyte-like phenotype and a unique ability to suppress T-cell responses. MDSCs have been shown to accumulate in cancer patients, but recent studies suggest that these cells are also present in humans and animals suffering from autoimmune diseases. We previously identified MDSCs in the synovial fluid (SF) of mice with experimental autoimmune arthritis. The goal of the present study was to identify MDSCs in the SF of patients with rheumatoid arthritis (RA).
Methods
RA SF cells were studied by flow cytometry using antibodies to MDSC cell surface markers as well as by analysis of cell morphology. The suppressor activity of RA SF cells toward autologous peripheral blood T cells was determined ex vivo. We employed both antigen-nonspecific (anti-CD3/CD28 antibodies) and antigen-specific (allogeneic cells) induction systems to test the effects of RA SF cells on the proliferation of autologous T cells.
Results
SF from RA patients contained MDSC-like cells, the majority of which showed granulocyte (neutrophil)-like phenotype and morphology. RA SF cells significantly suppressed the proliferation of anti-CD3/CD28-stimulated autologous T cells upon co-culture. When compared side by side, RA SF cells had a more profound inhibitory effect on the alloantigen-induced than the anti-CD3/CD28-induced proliferation of autologous T cells.
Conclusion
MDSCs are present among RA SF cells that are commonly regarded as inflammatory neutrophils. Our results suggest that the presence of neutrophil-like MDSCs in the SF is likely beneficial, as these cells have the ability to limit the expansion of joint-infiltrating T cells in RA.
doi:10.1186/1471-2474-15-281
PMCID: PMC4152562  PMID: 25138129
Rheumatoid arthritis; Myeloid-derived suppressor cells; T cells; Synovial fluid
14.  Very small size proteoliposomes abrogate cross-presentation of tumor antigens by myeloid-derived suppressor cells and induce their differentiation to dendritic cells 
Background
Myeloid-derived suppressor cells (MDSCs) are among the major obstacles that adjuvants for cancer vaccines have to overcome. These cells cross-present tumor-associated antigens (TAA) to naive T lymphocytes with a tolerogenic outcome. Very Small Size Proteoliposomes (VSSP) is used as adjuvant by four therapeutic cancer vaccines currently in Phase I and II clinical trials. We previously found that VSSP reduces the suppressive function of MDSCs, then activating antigen-specific CTL responses in tumor-bearing (TB) mice, with the consequent reduction of tumor growth. However the mechanistic explanation for the immunomodulatory effect of this adjuvant in TB hosts has not been addressed before.
Methods
TB mice were inoculated with VSSP and MDSCs isolated and characterized by their expression of Arg1 and Nos2 genes by RT-PCR. The effect of VSSP on antigen cross-presentation by MDSCs, regulatory T cells (Tregs) expansion and MDSCs differentiation towards dendritic cells (DCs) was analyzed by FACS. Student’s t test or ANOVA and Tukey’s tests were used for statistical analyses.
Results
After inoculating VSSP into TB mice, a significant reduction of Arg1 and Nos2 gene expression was observed in recovered MDSCs. Concurrently the ability of these cells to induce down-regulation of CD3ζ chain on T cells was lost. Likewise in mice inoculated with the adjuvant lower percentages of Tregs were detected. In vitro, VSSP treatment was enough to differentiate MDSCs into phenotypically mature DCs, eliminating the former suppressive effect. Noteworthy, in vivo administration of VSSP to OVA-expressing (EG.7) TB mice abrogated this model antigen cross-presentation by splenic MDSCs. Similar results were obtained even when OVA antigen was administered into these TB mice formulated in VSSP. On the contrary, immunization with the same protein in polyI:C did not change the percentage of MDSCs expressing SIINFEKL/H-2Kb complexes, whereas a concomitant injection of VSSP aborted the limitations of polyI:C in this setting.
Conclusions
Altogether, these results indicate that VSSP has the peculiar capacity of inhibiting TAA cross-presentation and certain suppressive mechanisms on MDSCs which in turn, combined with the ability to induce differentiation of these cells into antigen-presenting cells (APCs), sustains this adjuvant as an ideal immunomodulator for cancer immunotherapy.
doi:10.1186/2051-1426-2-5
PMCID: PMC4019907  PMID: 24829762
Adjuvants; MDSCs; Tumors; Cross-presentation; APCs
15.  Dynamic Change and Impact of Myeloid Derived Suppressor Cells in Allogeneic Bone Marrow Transplantation in Mice 
Myeloid-derived suppressor cells (MDSCs) are a group of myeloid cells comprised of hematopoietic progenitor cells, immature macrophages, dendritic cells and granulocytes, which accumulate in inflammatory diseases and various cancers. Here, we investigated the dynamic changes and effects of MDSCs in graft-versus-host disease (GVHD) development and/or tumor relapse after syngeneic and allogeneic bone marrow transplantation (BMT). We found that adding functional MDSCs in donor graft alleviated GVHD, whereas removal of MDSCs in vivo exacerbated GVHD. Following T cell-deplete BMT, MDSCs transiently accumulated in the blood and spleen of recipients without GVHD; In contrast, after T cell-replete BMT, the levels of blood MDSCs were constantly elevated in recipients with GVHD. MDSC accumulation positively correlated with the severity of GVHD. Additionally, MDSC accumulation was further increased upon tumor relapse. Although MDSCs isolated from both syngeneic and allogeneic BMT recipients inhibited T-cell proliferation in response to alloantigen stimulation ex vivo, MDSCs from the recipients with GVHD showed much higher suppressive potency compared to those from recipients without GVHD. These results indicate that MDSCs can regulate the immune response in acute GVHD, and possibly tumor relapse, subsequent to allogeneic BMT.
doi:10.1016/j.bbmt.2013.01.008
PMCID: PMC4011929  PMID: 23376089
16.  Myeloid-derived suppressor cell accumulation and function in patients with newly diagnosed glioblastoma 
Neuro-Oncology  2011;13(6):591-599.
To assess the accumulation of myeloid-derived suppressor cells (MDSCs) in the peripheral blood of patients with glioma and to define their heterogeneity and their immunosuppressive function.
Peripheral blood mononuclear cells (PBMCs) from healthy control subjects and from patients with newly diagnosed glioma were stimulated with anti-CD3/anti-CD28 and T cells assessed for intracellular expression of interferon (IFN)–γ. Antibody staining of PBMCs from glioma patients and healthy donors (CD33, HLADR, CD15, and CD14) followed by 4-color flow cytometry analysis–defined MDSC levels in the peripheral blood. To assess the role of MDSCs in suppressing T cell IFNγ production, PBMCs were depleted of MDSCs using anti-CD33 and anti-CD15 antibody-coated beads prior to T cell stimulation. Enzyme-linked immunosorbent assays were used to assess plasma arginase activity and the level of granulocyte colony-stimulating factor (G-CSF).
Patients with glioblastoma have increased MDSC counts (CD33+HLADR−) in their blood that are composed of neutrophilic (CD15+; >60%), lineage-negative (CD15−CD14−; 31%), and monocytic (CD14+; 6%) subsets. After stimulation, T cells from patients with glioblastoma had suppressed IFN-γ production when compared with healthy, age-matched donor T cells. Removal of MDSCs from the PBMCs with anti-CD33/CD15–coated beads significantly restored T cell function. Significant increases in arginase activity and G-CSF levels were observed in plasma specimens obtained from patients with glioblastoma.
The accumulation of MDSCs in peripheral blood in patients with glioma likely promotes T cell immune suppression that is observed in this patient population. Increased plasma levels of arginase and G-CSF may relate to MDSC suppressor function and MDSC expansion, respectively, in patients with glioma.
doi:10.1093/neuonc/nor042
PMCID: PMC3107102  PMID: 21636707
Arginase; GBM; G-CSF; MDSC; neutrophil
17.  Myeloid-derived suppressor cells expand during breast cancer progression and promote tumor-induced bone destruction 
Oncoimmunology  2012;1(9):1484-1494.
Myeloid-derived suppressor cells (MDSCs), identified as Gr1+CD11b+ cells in mice, expand during cancer and promote tumor growth, recurrence and burden. However, little is known about their role in bone metastases. We hypothesized that MDSCs may contribute to tumor-induced bone disease, and inoculated breast cancer cells into the left cardiac ventricle of nude mice. Disease progression was monitored weekly by X-ray and fluorescence imaging and MDSCs expansion by fluorescence-activated cell sorting. To explore the contribution of MDSCs to bone metastasis, we co-injected mice with tumor cells or PBS into the left cardiac ventricle and Gr1+CD11b+ cells isolated from healthy or tumor-bearing mice into the left tibia. MDSCs didn’t induce bone resorption in normal mice, but increased resorption and tumor burden significantly in tumor-bearing mice. In vitro experiments showed that Gr1+CD11b+ cells isolated from normal and tumor-bearing mice differentiate into osteoclasts when cultured with RANK ligand and macrophage colony-stimulating factor, and that MDSCs from tumor-bearing mice upregulate parathyroid hormone-related protein (PTHrP) mRNA levels in cancer cells. PTHrP upregulation is likely due to the 2-fold increase in transforming growth factor β expression that we observed in MDSCs isolated from tumor-bearing mice. Importantly, using MDSCs isolated from GFP-expressing animals, we found that MDSCs differentiate into osteoclast-like cells in tumor-bearing mice as evidenced by the presence of GFP+TRAP+ cells. These results demonstrate that MDSCs expand in breast cancer bone metastases and induce bone destruction. Furthermore, our data strongly suggest that MDSCs are able to differentiate into osteoclasts in vivo and that this is stimulated in the presence of tumors.
doi:10.4161/onci.21990
PMCID: PMC3525604  PMID: 23264895
TGFβ; bone metastasis; breast cancer; myeloid-derived suppressor cells (MDSCs); osteoclasts PTHrP
18.  Proinflammatory S100 Proteins Regulate the Accumulation of Myeloid-Derived Suppressor Cells1 
Chronic inflammation is a complex process that promotes carcinogenesis and tumor progression; however, the mechanisms by which specific inflammatory mediators contribute to tumor growth remain unclear. We and others recently demonstrated that the inflammatory mediators IL-1β, IL-6, and PGE2 induce accumulation of myeloid-derived suppressor cells (MDSC) in tumor-bearing individuals. MDSC impair tumor immunity and thereby facilitate carcinogenesis and tumor progression by inhibiting T and NK cell activation, and by polarizing immunity toward a tumor-promoting type 2 phenotype. We now show that this population of immature myeloid cells induced by a given tumor share a common phenotype regardless of their in vivo location (bone marrow, spleen, blood, or tumor site), and that Gr1highCD11bhighF4/80−CD80+IL4Rα+/−Arginase+ MDSC are induced by the proinflammatory proteins S100A8/A9. S100A8/A9 proteins bind to carboxylated N-glycans expressed on the receptor for advanced glycation end-products and other cell surface glycoprotein receptors on MDSC, signal through the NF-κB pathway, and promote MDSC migration. MDSC also synthesize and secrete S100A8/A9 proteins that accumulate in the serum of tumor-bearing mice, and in vivo blocking of S100A8/A9 binding to MDSC using an anti-carboxylated glycan Ab reduces MDSC levels in blood and secondary lymphoid organs in mice with metastatic disease. Therefore, the S100 family of inflammatory mediators serves as an autocrine feedback loop that sustains accumulation of MDSC. Since S100A8/A9 activation of MDSC is through the NF-κB signaling pathway, drugs that target this pathway may reduce MDSC levels and be useful therapeutic agents in conjunction with active immunotherapy in cancer patients.
PMCID: PMC2810501  PMID: 18802069
19.  Myeloid-Derived Suppressor Cells in Multiple Myeloma: Pre-Clinical Research and Translational Opportunities 
Frontiers in Oncology  2014;4:348.
Immunosuppressive cells have been reported to play an important role in tumor-progression mainly because of their capability to promote immune-escape, angiogenesis, and metastasis. Among them, myeloid-derived suppressor cells (MDSCs) have been recently identified as immature myeloid cells, induced by tumor-associated inflammation, able to impair both innate and adaptive immunity. While murine MDSCs are usually identified by the expression of CD11b and Gr1, human MDSCs represent a more heterogeneous population characterized by the expression of CD33 and CD11b, low or no HLA-DR, and variable CD14 and CD15. In particular, the last two may alternatively identify monocyte-like or granulocyte-like MDSC subsets with different immunosuppressive properties. Recently, a substantial increase of MDSCs has been found in peripheral blood and bone marrow (BM) of multiple myeloma (MM) patients with a role in disease progression and/or drug resistance. Pre-clinical models recapitulating the complexity of the MM-related BM microenvironment (BMM) are major tools for the study of the interactions between MM cells and cells of the BMM (including MDSCs) and for the development of new agents targeting MM-associated immune-suppressive cells. This review will focus on current strategies for human MDSCs generation and investigation of their immunosuppressive function in vitro and in vivo, taking into account the relevant relationship occurring within the MM–BMM. We will then provide trends in MDSC-associated research and suggest potential application for the treatment of MM.
doi:10.3389/fonc.2014.00348
PMCID: PMC4258997  PMID: 25538892
MDSC; myeloma; immunosuppression; cancer; pre-clinical models
20.  Regulation of accumulation and function of myeloid derived suppressor cells in different murine models of hepatocellular carcinoma 
Journal of hepatology  2013;59(5):1007-1013.
Background and aims
Myeloid derived suppressor cells (MDSC) are immature myeloid cells with immunosuppressive activity. They accumulate in tumor-bearing mice and humans with different types of cancer, including hepatocellular carcinoma (HCC). The aim of this study was to examine the biology of MDSC in murine HCC models and to identify a model, which mimics the human disease.
Methods:
The comparative analysis of MDSC was performed in mice, bearing transplantable, diethylnitrosoamine (DEN)-induced and MYC-expressing HCC at different ages.
Results:
An accumulation of MDSC was found in mice with HCC irrespectively of the model tested. Transplantable tumors rapidly induced systemic recruitment of MDSC, in contrast to slow-growing DEN-induced or MYC-expressing HCC, where MDSC numbers only increased intra-hepatically in mice with advanced tumors. MDSC derived from mice with subcutaneous tumors were more suppressive than those from mice with DEN-induced HCC. Enhanced expression of genes associated with MDSC generation (GM-CSF, VEGF, IL-6, IL-1β) and migration (MCP-1, KC, S100A8, S100A9) was observed in mice with subcutaneous tumors. In contrast, only KC levels increased in mice with DEN-induced HCC. Both KC and GM-CSF over-expression or anti-KC and anti-GM-CSF treatment controlled MDSC frequency in mice with HCC. Finally, the frequency of MDSC decreased upon successful anti-tumor treatment with sorafenib.
Conclusions:
Our data indicate that MDSC accumulation is a late event during hepatocarcinogenesis and differs significantly depending on the tumor model studied.
doi:10.1016/j.jhep.2013.06.010
PMCID: PMC3805787  PMID: 23796475
21.  The Tumor-Promoting Flow of Cells Into, Within and Out of the Tumor Site: Regulation by the Inflammatory Axis of TNFα and Chemokines 
Cancer Microenvironment  2011;5(2):151-164.
Tumors are dynamic organs, in which active processes of cell motility affect disease course by regulating the composition of cells at the tumor site. While sub-populations of tumor-promoting leukocytes are recruited inward and endothelial cell migration stands in the basis of vascular branching throughout the tumor, cancer cells make their way out of the primary site towards specific metastatic sites. This review describes the independent and cross-regulatory roles of inflammatory chemokines and of the inflammatory cytokine tumor necrosis factor α (TNFα) in determining cell motility processes that eventually have profound effects on tumor growth and metastasis. First, the effects of inflammatory chemokines such as CCL2 (MCP-1), CCL5 (RANTES) and CXCL8 (IL-8) are described, regulating the inward flow of leukocyte sub-populations with pro-tumoral activities, such as tumor-associated macrophages (TAM), myeloid-derived suppressor cells (MDSC), tumor-associated neutrophils (TAN), Th17 cells and Tregs. Then, the ability of inflammatory chemokines to induce endothelial cell migration, sprouting and tube formation is discussed, with its implications on tumor angiogenesis. This part is followed by an in depth description of the manners by which TNFα potentiates the above activities of the inflammatory chemokines, alongside with its ability to directly induce migratory processes in the tumor cells thus promoting metastasis. Note worthy is the ability of TNFα to induce in the tumor cells the important process of epithelial-to-mesenchymal transition (EMT). Emphasis is given to the ability of TNFα to establish an inflammatory network with the chemokines, and in parallel to form a cell re-modeling network together with transforming growth factor β (TGFβ). The review concludes by discussing the implications of such networks on disease course, and on the future design of therapeutic measures in cancer.
doi:10.1007/s12307-011-0094-3
PMCID: PMC3399063  PMID: 22190050
Inflammation; Chemokines; Cytokines; Tumor necrosis factor α; Angiogenesis; Leukocytes
22.  Epigenetic silencing of retinoblastoma gene regulates pathologic differentiation of myeloid cells in cancer 
Nature immunology  2013;14(3):211-220.
Two major populations of myeloid-derived suppressor cells (MDSC), monocytic MDSC (M-MDSC) and polymorphonuclear MDSC (PMN-MDSC) regulate immune responses in cancer and other pathologic conditions. Under physiologic conditions, Ly6ChiLy6G− inflammatory monocytes, which are the normal counterpart of M-MDSC, differentiate into macrophages and dendritic cells (DCs). PMN-MDSC is the predominant group of MDSC that accumulates in cancer. Here we show that a large proportion of M-MDSC in tumor-bearing mice acquired phenotypic, morphological and functional features of PMN-MDSC. Acquisition of this phenotype, but not the functional attributes of PMN-MDSC, was mediated by transcriptional silencing of the retinoblastoma (Rb) gene through epigenetic modifications mediated by histone deacetylase 2 (HDAC-2). These data demonstrate novel mechanism regulation of myeloid cells in cancer.
doi:10.1038/ni.2526
PMCID: PMC3578019  PMID: 23354483
23.  HIF-1α regulates function and differentiation of myeloid-derived suppressor cells in the tumor microenvironment 
The Journal of Experimental Medicine  2010;207(11):2439-2453.
The hypoxic environment of tumors dictates the phenotype of local myeloid-derived suppressor cells (MDSCs) via HIF-1a expression; hypoxia converts splenic MDSCs from specific into nonspecific suppressors.
Myeloid-derived suppressor cells (MDSCs) are a major component of the immune-suppressive network described in cancer and many other pathological conditions. We demonstrate that although MDSCs from peripheral lymphoid organs and the tumor site share similar phenotype and morphology, these cells display profound functional differences. MDSC from peripheral lymphoid organs suppressed antigen-specific CD8+ T cells but failed to inhibit nonspecific T cell function. In sharp contrast, tumor MDSC suppressed both antigen-specific and nonspecific T cell activity. The tumor microenvironment caused rapid and dramatic up-regulation of arginase I and inducible nitric oxide synthase in MDSC, which was accompanied by down-regulation of nicotinamide adenine dinucleotide phosphate–oxidase and reactive oxygen species in these cells. In contrast to MDSC from the spleen, MDSC from the tumor site rapidly differentiated into macrophages. Exposure of spleen MDSC to hypoxia resulted in the conversion of these cells to nonspecific suppressors and their preferential differentiation to macrophages. Hypoxia-inducible factor (HIF) 1α was found to be primarily responsible for the observed effects of the tumor microenvironment on MDSC differentiation and function. Thus, hypoxia via HIF-1α dramatically alters the function of MDSC in the tumor microenvironment and redirects their differentiation toward tumor-associated macrophages, hence providing a mechanistic link between different myeloid suppressive cells in the tumor microenvironment.
doi:10.1084/jem.20100587
PMCID: PMC2964584  PMID: 20876310
24.  Rgs2 Mediates Pro-Angiogenic Function of Myeloid Derived Suppressor Cells in the Tumor Microenvironment via Upregulation of MCP-1 
PLoS ONE  2011;6(4):e18534.
Background
Tumor growth is intimately linked with stromal interactions. Myeloid derived suppressor cells (MDSCs) are dramatically elevated in cancer patients and tumor bearing mice. MDSCs modulate the tumor microenvironment through attenuating host immune response and increasing vascularization.
Methodology/Principal Findings
In searching for molecular mediators responsible for pro-tumor functions, we found that regulator of G protein signaling-2 (Rgs2) is highly increased in tumor-derived MDSCs compared to control MDSCs. We further demonstrate that hypoxia, a common feature associated with solid tumors, upregulates the gene expression. Genetic deletion of Rgs2 in mice resulted in a significant retardation of tumor growth, and the tumors exhibit decreased vascular density and increased cell death. Interestingly, deletion of Rgs2 in MDSCs completely abolished their tumor promoting function, suggesting that Rgs2 signaling in MDSCs is responsible for the tumor promoting function. Cytokine array profiling identified that Rgs2−/− tumor MDSCs produce less MCP-1, leading to decreased angiogenesis, which could be restored with addition of recombinant MCP-1.
Conclusion
Our data reveal Rgs2 as a critical regulator of the pro-angiogenic function of MDSCs in the tumor microenvironment, through regulating MCP-1 production.
doi:10.1371/journal.pone.0018534
PMCID: PMC3073977  PMID: 21494556
25.  Tumor-expressed iNOS controls induction of functional myeloid derived suppressor cells (MDSC) through modulation of VEGF release1 
Inducible nitric oxide synthase (iNOS) is a hallmark of chronic inflammation which is also overexpressed in melanoma and other cancers. While iNOS is a known effector of myeloid-derived suppressor cell (MDSC)-mediated immunosuppression, its pivotal position at the interface of inflammation and cancer also makes it an attractive candidate regulator of MDSC recruitment. We hypothesized that tumor-expressed iNOS controls MDSC accumulation and acquisition of suppressive activity in melanoma. CD11b+Gr1+ MDSC derived from mouse bone marrow cells cultured in the presence of MT-RET-1 mouse melanoma cells or conditioned supernatants expressed STAT3 and reactive oxygen species (ROS) and efficiently suppressed T cell proliferation. Inhibition of tumor-expressed iNOS with the small molecule inhibitor L-NIL blocked accumulation of STAT3/ROS-expressing MDSC, and abolished their suppressive function. Experiments with VEGF-depleting antibody and recombinant VEGF identified a key role for VEGF in the iNOS-dependent induction of MDSC. These findings were further validated in mice bearing transplantable MT-RET-1 melanoma, where L-NIL normalized elevated serum VEGF levels; downregulated activated STAT3 and ROS production in MDSC; and reversed tumor-mediated immunosuppression. These beneficial effects were not observed in iNOS “knockout” mice, suggesting L-NIL acts primarily on tumor-rather than host-expressed iNOS to regulate MDSC function. A significant decrease in tumor growth and a trend towards increased tumor-infiltrating CD8+ T cells was also observed in MT-RET transgenic mice bearing spontaneous tumors. These data suggest a critical role for tumor-expressed iNOS in the recruitment and induction of functional MDSC by modulation of tumor VEGF secretion and upregulation of STAT3 and ROS in MDSC.
doi:10.4049/jimmunol.1103553
PMCID: PMC3358566  PMID: 22529296

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