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Seediscussions,stats,andauthorprofilesforthispublicationat:https://www.researchgate.net/publication/221904480 Coordinatedregulationofmyeloidcellsby tumours.NatRevImmunol ArticleinNatureReviewsImmunology·March2012 ImpactFactor:34.99·DOI:10.1038/nri3175·Source:PubMed CITATIONS READS 818 176 3authors,including: DmitryGabrilovich S.Ostrand-Rosenberg WistarInstitute UniversityofMaryland,BaltimoreCounty 228PUBLICATIONS22,015CITATIONS 139PUBLICATIONS9,072CITATIONS SEEPROFILE SEEPROFILE Availablefrom:S.Ostrand-Rosenberg Retrievedon:16June2016 F O C U S O N t u m our i m m uno lo g y & i m m unotRh py E er V I EaW S Coordinated regulation of myeloid cells by tumours Dmitry I. Gabrilovich1, Suzanne Ostrand-Rosenberg2 and Vincenzo Bronte3 Abstract | Myeloid cells are the most abundant nucleated haematopoietic cells in the human body and are a collection of distinct cell populations with many diverse functions. The three groups of terminally differentiated myeloid cells — macrophages, dendritic cells and granulocytes — are essential for the normal function of both the innate and adaptive immune systems. Mounting evidence indicates that the tumour microenvironment alters myeloid cells and can convert them into potent immunosuppressive cells. Here, we consider myeloid cells as an intricately connected, complex, single system and we focus on how tumours manipulate the myeloid system to evade the host immune response. Myeloid-derived suppressor cells (MDSCs). A group of immature CD11b+GR1+ cells that includes precursors of macrophages, granulocytes, dendritic cells and myeloid cells. These cells are produced in response to various tumour-derived cytokines and have been shown to inhibit tumour-specific immune responses. H. Lee Moffitt Cancer Center and Research Institute, 12902 Magnolia Drive, Tampa, Florida 33647, USA. 2 University of Maryland Baltimore County, 1000 Hilltop Circle, Baltimore, Maryland 21250, USA. 3 Verona University, P. le L.A. Scuro 10, 37134 Verona, Italy. e-mails: dmitry.gabrilovich@ moffitt.org; [email protected]; [email protected] doi:10.1038/nri3175 1 Myeloid cells are the most abundant haematopoietic cells in the human body and have diverse functions. All myeloid cells arise from multipotent haematopoietic stem cells (HSCs) that develop into mature myeloid cells through sequential steps of differentiation. However, myeloid progenitors do not form a hierarchical system but can instead be considered as a network of cells that can differentiate into various subsets of more-specialized myeloid cells (FIG. 1). The three groups of terminally differentiated myeloid cells — macrophages, dendritic cells (DCs) and granulocytes — are essential for the normal functions of the innate and adaptive immune systems. Classically, these cells protect organisms from pathogens, eliminate dying cells and mediate tissue remodelling. Although the contribution of myeloid cells to tumour pathogenesis has been recognized for over 100 years, only during the past two decades has their crucial role in promoting tumour angiogenesis, cell invasion and metastasis been appreciated (reviewed in REFS 1–3). Mast cells have also been implicated in the regulation of tumour progression (reviewed in REF. 4). Mounting evidence indicates that the tumour microenvironment alters myeloid cells by converting them into potent immunosuppressive cells. In recent years the concept of myeloid-derived suppressor cells (MDSCs) (described below) has emerged. However, the wealth of new information concerning myeloid cells in cancer has also produced confusion. In most studies, individual myeloid cell populations have been examined independently, generating fragmented information that has contributed to a disjointed view of the role of myeloid cells in immune responses in cancer. In addition, their overlapping expression of cell-surface markers has made it difficult to distinguish between different myeloid cell populations, further obscuring the nature of specific myeloid cell subsets in cancer. These complications limit our understanding of myeloid cell biology and hamper attempts to develop and optimize therapeutic interventions. In this Review, we present a cohesive view of the effects of tumours on myeloid cells. Our goal is not to provide a comprehensive overview of the changes in individual populations of myeloid cells, as this has been accomplished in other recent reviews. Instead, we briefly summarize the effects that tumours have on terminally differentiated myeloid cell subsets and then focus on discussing myeloid cell interactions and responses during tumour development as an intricately connected, single (albeit complex) system. Dendritic cells DCs are terminally differentiated myeloid cells that specialize in antigen processing and presentation. DCs differentiate in the bone marrow from various progenitors5–8. They can also differentiate from monocytes under certain conditions, although most DCs in mouse lymphoid organs are not derived from monocytes5,9. By contrast, monocytes are the major precursors of DCs in humans10. Two major subsets of DCs are currently recognized: conventional DCs (cDCs) and plasmacytoid DCs (pDCs). Although these cells share some common progenitors, their differentiation is controlled by distinct genetic programmes and they have different morphologies, markers and functions11 (TABLE 1). The centrepiece of DC biology is the concept of functional NATURE REVIEWS | IMMUNOLOGY VOLUME 12 | APRIL 2012 | 253 © 2012 Macmillan Publishers Limited. All rights reserved REVIEWS Lymphoid cells HSC T cell CLP B cell MPP CMLP NK cell Megakaryocyte Granulocytes MEP GMP CMP Basophil Eosinsophil Erythrocytes MCP MDP Mast cell Monocyte Macrophage CDP cDC Neutrophil pDC Figure 1 | Myeloid cell differentiation under normal physiological conditions. Myeloid cells originate from haematopoietic stem cells (HSCs) multipotent Natureand Reviews | Immunology progenitor cells (MPPs). The figure illustrates the network of progenitor cells that gives rise to the various haematopoietic cell lineages. cDC, conventional DC; CDP, common DC progenitor; CLP, common lymphoid progenitor; CMLP, common myelolymphoid progenitor; CMP, common myeloid progenitor; DC, dendritic cell; GMP, granulocyte and macrophage progenitor; MCP, mast cell progenitor; MDP, macrophage and DC progenitor; MEP, megakaryocyte and erythroid progenitor; NK, natural killer; pDC, plasmacytoid DC. Pathogen-associated molecular patterns (PAMPs). These are molecular motifs that are found in pathogens but not in mammalian cells. Examples include terminally mannosylated and polymannosylated compounds, which bind to the mannose receptor CD206, and various microbial products that activate host Toll-like receptors, such as bacterial lipopolysaccharides, hypomethylated DNA, flagellin and double-stranded RNA. activation and maturation in response to ‘dangerous’ stimuli. Differentiated DCs reside in tissues as ‘immature’ cells that actively take up tissue antigens but are poor antigen presenters and do not promote effector T cell differentiation. Only functionally activated DCs can effectively stimulate immune responses. DCs are activated in response to stimuli associated with bacteria, viruses or damaged tissues; such stimuli are commonly referred to as pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs). The activation of DCs leads to profound changes in their gene expression, resulting in increased expression of co-stimulatory molecules and cytokines that promote T cell activation and also in the upregulation of chemokine receptors that drive the migration of DCs to lymphoid tissues. pDCs constitute a minor population of DCs; they have a morphology reminiscent of plasma cells, express Toll-like receptor 7 (TLR7) and TLR9 (this receptor is not expressed by human cDCs) and produce large amounts of interferon-α (IFNα) in response to the activation of their TLRs by viral nucleic acids or self DNA11. A more detailed discussion of DC biology can be found in recent reviews9,12. Effects of cancer on DCs. That cancer can have profound effects on the function of DCs has been known for more than 20 years. It is well established that DCs in tumourbearing hosts do not adequately stimulate an immune response, and this potentially contributes to tumour evasion of immune recognition. Evidence from numerous studies strongly indicates that abnormal myelopoiesis is the dominant mechanism responsible for DC defects in cancer 13. This abnormal differentiation produces at least three main results: decreased production of mature functionally competent DCs; increased accumulation of immature DCs at the tumour site; and increased production of immature myeloid cells13. In recent years, multiple clinical studies have confirmed the findings of earlier studies and have indicated that there is a decreased presence and defective functionality of mature DCs in patients with breast 14, non-small cell lung 15, pancreatic16, cervical17, hepatocellular 18 or prostate cancer, or glioma19. In addition to the many tumour-derived soluble factors previously implicated in abnormal DC differentiation, such as vascular endothelial growth factor (VEGF), macrophage colony-stimulating factor (M-CSF) and interleukin‑6 (IL‑6), recent studies have shown that other factors present in the tumour microenvironment impair normal DC functions. The tumour microenvironment is predominantly characterized by hypoxia, accumulation of extracellular adenosine, increased levels of lactate and a decreased pH. DC migration and function are severely impaired by hypoxia and adenosine20,21. The transcription factor hypoxia-inducible factor 1α (HIF1α) is upregulated by DCs in the hypoxic tumour environment and was shown to induce the expression of adenosine receptor A2B by human DCs, causing these DCs to drive the development of TH2 cells (T helper 2 cells) rather than that of TH1 cells, which have more-potent antitumour activity 22. DCs differentiated in the presence of adenosine had impaired allostimulatory activity in a mixed leukocyte reaction, and they expressed higher levels of the pro-angiogenic cytokine VEGF, the pro-inflammatory cytokines IL‑6 and IL‑8, and the immunosuppressive mediators IL‑10, cyclooxygenase 2 (COX2), transforming growth factor-β (TGFβ) and indoleamine 2,3‑dioxygenase (IDO)23. Addition of lactic acid during DC differentiation in vitro also induced a phenotype comparable with that of tumour-associated DCs. Blockade of lactic acid production reverted the tumour-induced DC phenotype to normal24. DCs found in the peripheral blood and lymphoid organs of tumour-bearing mice or of patients with cancer, and especially DCs closely associated with the tumour, have an increased accumulation of lipids. This is mediated primarily through upregulation of macrophage scavenger receptor types I and II, and it impairs the ability of DCs to process soluble proteins and stimulate tumour-specific T cell responses25. Some DCs in tumour-bearing hosts actively suppress T cell function, and both phenotypically immature and phenotypically mature DCs may be conditioned by the environment to support immune tolerance or immuno suppression 26,27. MHC-II +CD11b +CD11c + tumourinfiltrating mouse DCs have been shown to suppress CD8+ T cells and antitumour immune responses through 254 | APRIL 2012 | VOLUME 12 www.nature.com/reviews/immunol © 2012 Macmillan Publishers Limited. All rights reserved F O C U S O N t u m our i m m uno lo g y & i m m unotRh py E er V I EaW S Table 1 | Phenotypic definitions used for isolating different myeloid cell populations Damage-associated molecular patterns (DAMPs). As a result of cellular stress, cellular damage and non-physiological cell death, DAMPs are released from the degraded stroma (in the case of hyaluronate, for example), from the nucleus (in the case of high-mobility group box 1 protein (HMGB1), for example) and from the cytoplasm (for example, in the case of ATP, uric acid, S100 calcium-binding proteins and heat-shock proteins). Such host-derived DAMPs are thought to promote local inflammatory reactions. TH2 cells (T helper 2 cells). CD4 T cells are classified on the basis of the types of effector cytokine that they secrete. TH2 cells produce interleukin‑4 (IL‑4), IL‑5 and IL‑13, and they support humoral immunity and downregulate TH1 cell responses. + Tissue Cell population Subpopulations Phenotype Mouse lymphoid organs DCs ND CD11c+F4/80–GR1–MHC-II+ Conventional DCs* CD11c+CD11b+MHC-II+CD205+F4/80–GR1–CD115low Plasmacytoid DCs CD11c+CD11b–B220+SIGLEC-H+GR1+F4/80– ND CD11b+LY6C+LY6G–CD11c–CD115+ Resident monocytes CD11b+LY6ClowLY6G–CD115+MHC-II–F4/80hiCD11c– Inflammatory monocytes CD11b+LY6ChiLY6G–CD115+MHC-II–F4/80+CD11c– ND F4/80+CD11b+GR1– M1 macrophages iNOS+IL‑12+CD86+MHC-IIhi M2 macrophages CD206+CD163+CD36+ARG1+MHC-IIlowIL‑10+IL-4Rα+ FIZZ1+YM1+ Granulocytes Various CD11b+LY6G+LY6ClowF4/80–CD11c– MDSCs ND CD11b+GR1+CD11c–F4/80+/–CD124+ Polymorphonuclear MDSCs CD11b+GR1hiLY6ClowLY6G+CD49d– Monocytic MDSCs CD11b+GR1midLY6ChiLY6G–CD49d+ ND LIN–HLA-DR+BDCA1+CD209+ Monocytes Macrophages Mouse tumours Human blood TH1 cells (T helper 1 cells). TH1 cells produce interferon‑γ, lymphotoxin‑α and tumour necrosis factor, and they support cell-mediated immunity. An imbalance between TH1 cell responses and TH2 cell responses is thought to contribute to the pathogenesis of various infections, allergic responses and autoimmune diseases. Mixed leukocyte reaction A tissue-culture technique for testing T cell reactivity and antigen-presenting cell (APC) activity. A population of T cells is cultured with MHC-mismatched APCs, and the proliferation of the T cells is determined by measuring the incorporation of 3 H-thymidine into the DNA of dividing cells. Indoleamine 2,3‑dioxygenase (IDO). An intracellular haem-containing enzyme that catalyses the oxidative catabolism of tryptophan. IDO suppresses T cell responses and promotes immune tolerance in mammalian pregnancy, tumour resistance, chronic infection, autoimmunity and allergic inflammation. Human tumours DCs‡ Conventional DCs LIN–CD11c+CD11b+CD33+BDCA1+BDCA3+DC-SIGN+ Plasmacytoid DCs LIN–CD123+BDCA2+BDCA4+ Monocytes‡ ND CD14+HLA-DR+CD15– Macrophages ND CD14+CD68+ M1 macrophages iNOS+IL‑12+CD86+HLA-DR+ M2 macrophages CD206+CD163+CD36+HLA-DRlowIL‑10+CD124+ Granulocytes|| ND CD15+CD14–CD66b+CD16+ MDSCs ND LIN–CD11b+HLA-DR–CD33+ Polymorphonuclear MDSCs In addition to the above MDSC phenotype, these cells express CD15 and/or CD66b Monocytic MDSCs CD33+CD14+HLA-DRlow/– § ¶ ARG1, arginase 1; BDCA1, blood DC antigen 1 (also known as CD1c); BDCA2, blood DC antigen 2 (also known as CD303); BDCA3, blood DC antigen 3 (also known as CD141); BDCA4, blood DC antigen 4 (also known as CD304); DC, dendritic cell; DC-SIGN, DC-specific ICAM3-grabbing non-integrin; IL, interleukin; iNOS, inducible nitric oxide synthase; LIN, lineage markers (a lineage cocktail of antibodies specific for CD3, CD14, CD19 and CD56 is used to identify other leukocyte populations in the sample); MHC-II, MHC class II; MDSC, myeloid-derived suppressor cell; ND, not determined; SIGLEC-H, sialic acid-binding immunoglobulin-like lectin H. *Expression of 33D1 antigen (also known as DCIR2) or of DEC205 (also known as CD205) is specific for mouse DCs, but these markers are not expressed by all conventional DCs. ‡The mononuclear fraction is separated on a standard Ficoll gradient. §Expression of DEC205 is specific for human DCs but this marker is not expressed on all conventional DCs. || These cells are usually not present in the mononuclear fraction and require sedimentation after the removal of mononuclear cells. ¶ These cells are purified on a standard Ficoll gradient for the isolation of mononuclear cells. arginase 1 (ARG1) production28, an immunosuppressive mechanism previously attributed only to mouse tumourassociated macrophages (TAMs) and MDSCs (see below). Interestingly, pDCs infiltrating prostate tumours also use ARG1 and IDO to alter the functions of intratumoural CD8+ T cells, suggesting that immunosuppressive programmes might be shared across different myeloid cells in cancer 29. Human lung tumour cells can convert mature DCs into TGFβ-producing cells30, and mouse lung cancer can drive DCs to express high levels of IL‑10, nitric oxide, VEGF and ARG1 (REF. 31). The accumulation of IDO-expressing DCs (most of which are pDCs) in tumour-bearing mice and in some patients with cancer 32,33 provides another possible mechanism of immune suppression, as IDO activity limits T cell growth by depleting l‑tryptophan and also promotes T cell apoptosis by generating l‑tryptophan metabolites and by altering redox potentials through consumption of superoxide radicals. Evidence supports the hypothesis that IDO-expressing DCs enhance the suppressive abilities of forkhead box P3 (FOXP3)-expressing regulatory T cells (TReg cells) in certain settings of chronic inflammation34. As mentioned above, such immunosuppressive activities are primarily associated with DCs localized in tumour sites. However, abnormal DC differentiation and defective DC function is a systemic phenomenon that affects the myeloid cell lineage during cancer, as described in more detail below. Macrophages Macrophages are a group of terminally differentiated myeloid cells that are closely related to DCs. They are tissue-resident cells derived from monocytes circulating in peripheral blood. The macrophage population NATURE REVIEWS | IMMUNOLOGY VOLUME 12 | APRIL 2012 | 255 © 2012 Macmillan Publishers Limited. All rights reserved REVIEWS Tumour TGFβ PGE2, COX2, TGFβ TH2 cell TReg cell IL-4 IL-13 Resulting TAM phenotype IL-10hi IL-12low EGF+ IL-10 CD206hi CD163hi MHC-IIlow CCL18+ LPS-nonresponsive Hypoxia B cell TNF, PGE2 COX2 IL-23 IL-10 YM1+ FIZZ1+ IFNβ+ CCL5+ IL-10low CD68+ Inflammation IL-10hi IL-12low CD206hi MHC-IIlow MMP9+ IL-1β+ CCL5+ CCL22+ MMP7+ CD163+ CCL2, plasminogen TIE2+ Tumour-associated macrophage (TAM) IL-6+ PDL1+ CD11b+ F4/80+ FIZZ1+ YM1+ CD68+ Recruitment of macrophages to the tumour S100A8, S100A9 MDSC IL-10, TGFβ IL-12low CD163+ CD1d+ CD206+ IL-4Rα+ IL-12low IL-10hi TGFβ+ Figure 2 | The tumour microenvironment polarizes macrophages towards a tumour-promoting phenotype. Tumour cells produce factors that drive the generation of multiple types of regulatory immune cells, including T helper 2 (TH2) cells, regulatory T (TReg) cells, B cells and myeloid-derived suppressor cells (MDSCs). Tumour cells also modify their Reviews | Immunology local microenvironment to promote hypoxia and inflammation. Cells in the modified tumourNature microenvironment subsequently produce cytokines, chemokines and other molecules that promote the development of immunosuppressive tumour-associated macrophages (TAMs). Local hypoxia promotes macrophage expression of the angiopoietin receptor TIE2, which downregulates antitumour functions in macrophages. Cytokines produced by regulatory immune cells inhibit the antitumour response by suppressing macrophage expression of MHC class II molecules and interleukin‑12 (IL‑12), and by promoting macrophage expression of the anti-inflammatory cytokine IL‑10. Chemokines, such as CC‑chemokine ligand 2 (CCL2) and CCL5, promote further TAM development by enhancing the recruitment of macrophages to the tumour site. Factors derived from the tumour itself, such as tumour necrosis factor (TNF) and prostaglandin E2 (PGE2), also directly polarize macrophages. Crosstalk between tumour cells and MDSCs and between tumour cells and pro-inflammatory factors in the tumour microenvironment further amplifies the antitumour effects. See the main text for references and for a more detailed discussion of the molecules and cellular interactions that have been described for mouse macrophages and for human macrophages in the tumour environment. COX2, cyclooxygenase 2; EGF, epidermal growth factor; IFNβ, interferon-β; IL‑4Rα, IL‑4 receptor α-chain; LPS, lipopolysaccharide; MMP, matrix metalloproteinase; PDL1, PD1 ligand 1; TGFβ, transforming growth factor-β. Regulatory T cells (TReg cells). A specialized subset of CD4+ T cells that can suppress both innate and adaptive immune responses. These cells provide a crucial mechanism for the maintenance of peripheral self-tolerance, but may also limit the effectiveness of antitumour immune responses. includes a broad range of cells, the markers and functions of which reflect their tissue microenvironment (TABLE 1) . Their function in healthy individuals is to eliminate infectious agents, promote wound healing and regulate adaptive immunity (reviewed in REF. 35). The terminology ‘M1 macrophage’ and ‘M2 macrophage’ was coined to describe the different functional states of macrophages and was originally based on studies of mouse macrophages36. M1 or ‘classically activated’ macrophages are activated by IFNγ and bacterial products, express high levels of IL‑12 and low levels of IL‑10, and are tumoricidal. By contrast, M2 or ‘alternatively activated’ macrophages are activated by IL‑4, IL‑10, IL‑13 and glucocorticoid hormones, express high levels of IL‑10 and low levels of IL‑12, and facilitate tumour progression. As discussed by Mantovani37, the M1/M2 nomenclature is useful but is oversimplified because macrophages form a continuum of phenotypes. Although there are some differences between M2‑like mouse and human macrophages, phenotypically and functionally the macrophages in these two species are quite similar (TABLE 1). The role of macrophages in promoting tumorigenesis. There is extensive literature demonstrating that, in both mice and humans, macrophages are co-opted during malignancy to facilitate tumour growth (reviewed in REFS 1,2,37,38) (FIG. 2). Their presence is associated with poor clinical outcome1,38, and their pivotal role in cancer was recently highlighted by the demonstration that TAMs with a specific gene signature are associated with primary treatment failure in patients with Hodgkin’s lymphoma39. TAMs are M2‑like macrophages, and they mediate their effects via both non-immune and 256 | APRIL 2012 | VOLUME 12 www.nature.com/reviews/immunol © 2012 Macmillan Publishers Limited. All rights reserved F O C U S O N t u m our i m m uno lo g y & i m m unotRh py E er V I EaW S Plasminogen Plasminogen is the inactive precursor of plasmin, a serine protease involved in the dissolution of fibrin blood clots. A causal role has been suggested for plasmin generation in cancer cell invasion through extracellular matrix remodelling. Invariant NKT cells (Invariant natural killer T cells). A type of lymphocyte thought to be particularly important in bridging innate and adaptive immunity. These cells express a particular T cell receptor variable gene segment (Vα14 in mice and Vα24 in humans) that is precisely linked to a particular joining (Jα) gene segment. Typically, NKT cells co-express cell-surface markers encoded by the natural killer locus, and are activated by recognition of CD1d. immune mechanisms. Their non-immune mechanisms include the promotion of angiogenesis40, the facilitation of tumour cell invasion and metastasis41, and the protection of tumour cells from chemotherapy-induced apoptosis42 (see also reviews in REFS 1,2,37,38,43). TAMs sabotage antitumour immunity by eliminating M1 macrophage-mediated innate immune responses and by impairing T cell activation. Studies using transgenic mice have showed that IL‑12 produced by M1 macrophages promotes the activation of natural killer (NK) cells and TH1 cells, which facilitate the activation of cytotoxic T lymphocytes (CTLs). However, because TAMs do not produce IL‑12, they do not contribute to the activation of NK cells and T H1 cells. Instead, they produce IL‑10 and drive the development of TH2 cells. TH2 cells do not support the development of CTL responses, and their production of IL‑4 drives the development of TAMs44. In addition, IL‑10 that is produced by macrophages in the inflamed intestinal lamina propria is required to maintain TReg cell activity and prevent autoimmune colitis45, raising the possibility that TAM-derived IL‑10 may also promote tumour progression by enhancing TReg cell activity. TAMs are ineffective antigen-presenting cells, and they produce CC‑chemokine ligand 22 (CCL22), which attracts TReg cells that inhibit T cell activation46. Secretion of prostaglandin E2 (PGE2) and TGFβ 47 by TAMs further contributes to immune suppression. TAMs can also cause T cell apoptosis through their expression of PD1 ligand 1 (PDL1), which binds to its receptor programmed cell death protein 1 (PD1) on activated T cells48, and mouse macrophages produce ARG1, which deprives T cells of the l‑arginine that is necessary for their growth49. A similar mechanism is also used by MDSCs (see below). Inflammation is important for the recruitment of macrophages to tumour sites, and the pro-inflammatory mediators CCL2 (REF. 50) and plasminogen51,52 have essential roles in this process. TAM polarization. Because different areas of solid tumours have distinct microenvironments, the TAMs found within an individual tumour vary in phenotype and function. Seven subsets of TAMs have been identified in mouse mammary carcinoma and lung adenocarcinoma based on their expression of LY6C, MHC class II molecules, CX 3C‑chemokine receptor 1 (CX3CR1), CC‑chemokine receptor 2 (CCR2) and CD62L (also known as L‑selectin). These subsets have different half-lives, and their relative frequencies change as the tumour microenvironment evolves with disease progression53. Pro-angiogenic TAMs may express the angiopoietin receptor TIE2 (REF. 54) and/or have low expression levels of MHC class II molecules, and they localize to hypoxic regions 53. TAMs that promote early tumour cell invasion are enriched for WNT7B, a protein that is involved in normal developmental and repair responses55. Tumour-derived TGFβ and PGE2 promote the differentiation of macrophages that express high levels of GR1 and low levels of markers associated with M1‑type macrophages47. T cells have an important role in macrophage regulation during tumorigenesis. In a mouse model of breast cancer driven by transgenic expression of the polyomavirus middle T antigen, mice with mammary adenocarcinomas developed TH2 cells that produced IL‑4, which polarized TAMs to an M2 phenotype. These TAMs produced epidermal growth factor (EGF), which initiates tumour cell invasion, migration and metastasis by signalling through the corresponding receptor on malignant mammary epithelial cells44. TReg cells also regulate macrophages by orchestrating monocyte differentiation. A population of human CD4+CD25+CD127lowFOXP3+ TReg cells was shown to induce monocytes to differentiate into M2 macrophages by inhibiting their responsiveness to lipopolysaccharide (LPS), which induces M1 polarization, and by increasing their expression of the mannose receptor CD206 and the scavenger receptor CD163. TReg cell production of IL‑10, IL‑4 and IL‑13 promotes the non-responsiveness of macrophages to LPS56. In contrast to TReg cells, Vα24‑invariant NKT cells show cytotoxic activity towards TAMs and facilitate tumour rejection57. B cells also polarize macrophages towards a tumourpromoting phenotype58. A recent study in a mouse B16 melanoma model has shown that B cells decrease the production of tumour necrosis factor (TNF), IL‑1β and CCL3 by macrophages, whereas they increase macrophage production of IL‑10. This facilitates protumour macrophage activity as well as the synthesis of the M2‑associated markers YM1 (also known as CHI3L3) and FIZZ1 (also known as RELMα)59. In another report, autoantibodies polarized leukocytes, including macrophages, towards a tumour-promoting phenotype by interacting with activating Fc receptors on the leukocytes60. Polarization of macrophages towards an M2 phenotype is also mediated directly by tumour cells. For example, human ovarian cancer cells cause macrophages to produce increased levels of IL‑10, IL‑1β, CCL5, CCL22, matrix metalloproteinase 7 (MMP7), MMP9, CD206 and CD163. Tumour cell-derived TNF was partially responsible for this polarization through its induction of macrophage scavenger receptor types I and II62. Granulocytes Granulocytes are myeloid cells that are characterized by the presence of cytoplasmic granules and a specific nuclear morphology. The most abundant type of granulocytes in the body is neutrophils, which are also commonly referred to as polymorphonuclear leukocytes owing to their polylobed nuclei. Neutrophils possess a complex machinery to engulf and destroy bacteria. Neutrophils are not released from the bone marrow until they reach full maturity, but during inflammation neutrophil precursors (myelocytes and promyelocytes) can be released62. Human tumours can be infiltrated by mature granulocytes, the numbers of which can be independent prognostic factors for tumour recurrence63–66. Recent evidence has linked granulocytes, and particularly neutrophils, with tumour angiogenesis and metastasis, and NATURE REVIEWS | IMMUNOLOGY VOLUME 12 | APRIL 2012 | 257 © 2012 Macmillan Publishers Limited. All rights reserved REVIEWS has provided initial clues about the immunoregulatory roles of these cells in cancer. Tumour cells and tumourassociated stromal cells produce neutrophil-attracting CXC-chemokines and prokineticin 2 (the mammalian orthologue of BV8)67,68. In the lungs, tumour-derived granulocyte colony-stimulating factor (G‑CSF) also mobilizes granulocytes to pre-metastatic niches and supports subsequent metastasis formation, whereas prokineticin 2 aids tumour cell migration through Tumour environment HSC Basophil Aberrant differentiation of myeloid cells in tumour environment DC Eosinsophil Immature myeloid progenitor cells Monocyte Neutrophil PMN-MDSC Antigen-specific T cell tolerance and nonspecific suppression the activation of prokineticin receptor 1 (REF. 69). It is likely that granulocytes facilitate the angiogenic switch by expressing MMP9, which promotes tumour angio genesis by inducing VEGF expression in neoplastic tissue70. Mobilized granulocytes also release elastase, which then enters endosomal compartments of neoplastic cells and degrades insulin receptor substrate 1 (IRS1). Degradation of IRS1 facilitates interactions between phosphoinositide 3‑kinase (PI3K) and the receptor for the mitogen platelet-derived growth factor (PDGF), thus promoting tumour cell proliferation71. In contrast to these observations, a recent study demonstrated that, in mice bearing 4T1 breast tumours, neutrophils inhibited the formation of tumour meta stases through direct antitumour effects mediated by reactive oxygen species (ROS)72. These new data revisit the old concept of tumour-cytotoxic neutrophils and suggest a possible dichotomic polarization of neutrophils. Similarly to macrophages, neutrophils have been shown to shift from an antitumoural ‘N1’ phenotype to a pro-tumoural ‘N2’ phenotype in the cancer environment 73. TGFβ drives the N2 phenotype, and TGFβ blockade promotes an N1 phenotype with antitumour activity. In lung adenocarcinoma and mesothelioma models, TGFβ induces tumour-infiltrating neutrophils to develop a pro-tumoural phenotype, which is characterized by ARG1 expression and low levels of TNF, CCL3 and intercellular adhesion molecule 1 (ICAM1). In tumour-bearing animals, depletion of N2 neutrophils led to an increase in CD8+ T cell activity 73. In line with these findings, serum amyloid A1 protein induced the expansion of IL‑10‑secreting neutrophil populations that were able to suppress the antigen-specific proliferation of CD8+ T cells in human melanomas74. However, IL‑10 production by activated human neutrophils was not confirmed in a subsequent study 75. Myeloid cells as a single integrated system Neoplastic cells condition distant sites, such as the bone marrow and spleen, by releasing soluble factors that drive the accumulation of myeloid cells; these myeloid cells subsequently promote neovasculariza• Non-specific suppression tion and metastasis. This creates a tumour-driven • Produce cytokines and ‘macroenvironment’. As discussed above, this macro soluble factors that support environment conditions DCs, macrophages and tumour angiogenesis Suppressive DC TAM granulocytes to become immunosuppressive. However, the most prominent effect is the accumulation of highly immunosuppressive, immature myeloid cells. These cells were named MDSCs to highlight their comFigure 3 | Changes that occur in myeloid cells in cancer. Factors produced in the mon myeloid origin and immunoregulatory proper76 tumour microenvironment by tumour cells and stromal cellsNature promote the aberrant Reviews | Immunology ties (TABLE 1). Immature myeloid cells with the same differentiation of myeloid lineage cells. The dotted lines show the normal pathways phenotype as MDSCs are continually generated in the of myeloid cell differentiation from immature myeloid precursor cells to dendritic bone marrow of healthy individuals and differentiate cells (DCs), macrophages and granulocytes, as depicted in FIG. 1. The solid bold lines into mature myeloid cells without causing detectable indicate the aberrant pathways of myeloid cell differentiation that occur in cancer, immunosuppression. However, in cancer, myeloid cell in which the tumour environment can promote the development of various differentiation is diverted from its normal pathway — immunosuppressive populations, including monocytic myeloid-derived suppressor cells (M-MDSCs), polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs), which leads to the terminal differentiation of mature macrophages, DCs and granulocytes — towards a suppressive DCs and tumour-associated macrophages (TAMs). The dotted thick line pathway that favours the differentiation of pathological depicts a pathway of cell differentiation that has been suggested but has not yet been MDSCs (FIG. 3). confirmed. HSC, haematopoietic stem cell. Macrophage M-MDSC 258 | APRIL 2012 | VOLUME 12 www.nature.com/reviews/immunol © 2012 Macmillan Publishers Limited. All rights reserved F O C U S O N t u m our i m m uno lo g y & i m m unotRh py E er V I EaW S Characteristics of MDSCs. MDSCs were originally identified in tumour-bearing mice as cells that co-express CD11b and GR1; however, their phenotype in cancer is rather diverse77,78. Currently, two main MDSC populations have been characterized: monocytic MDSCs and polymorphonuclear MDSCs (also known as granulocytic MDSCs) (TABLE 1). In tumour-bearing mice, the polymorphonuclear MDSC subset is the prevalent population of MDSCs. Polymorphonuclear MDSCs suppress antigenspecific CD8+ T cells predominantly by producing ROS. Polymorphonuclear MDSCs represent the major subset of circulating MDSCs; however, they are less immuno suppressive than monocytic MDSCs when assessed on a per cell basis79–81. In human studies, the number of monocytic MDSCs, but not of polymorphonuclear MDSCs, correlated directly with suppression of in vitro T cell activation82. In addition to their specific markers (TABLE 1), monocytic MDSCs express varying levels of classic monocyte markers, such as F4/80 (also known as EMR1), CD115 (also known as M‑CSFR), 7/4 (also known as LY6B) and CCR2 (REFS 79–81,83). They suppress CD8+ T cells predominantly via expression of the enzymes ARG1 and inducible nitric oxide synthase (iNOS) and through the production of reactive nitrogen species79–81. This subset of MDSCs may also include progenitors that give rise to a subset of CD11bhiGR1lowLY6G–F4/80hiMHC-II+ macrophages with potent immunosuppressive properties81,84–86. MDSCs with the phenotype LIN – HLA-DR – CD33+CD11b+ have been isolated from the blood of patients with glioblastoma, breast cancer, colon cancer, lung cancer or kidney cancer 78,87–90. These cells share features and properties with progranulocytes89. The frequency of this immature cell population may reflect the tumour burden, and a high frequency correlates with a poor prognosis and radiographic progression in patients with breast or colorectal cancer 88,89,91. In addition, the frequency of each MDSC subset appears to be influenced by the type of cancer. Patients with renal cancer have immunosuppressive CD11b+CD14– CD15+CD66b+VEGFR1+ polymorphonuclear MDSCs92, whereas CD14+CD11b+HLA-DRlow/– monocytic MDSCs circulate in the blood of patients with melanoma, multiple myeloma, prostate cancer, hepatocellular carcinoma or head and neck cancer 82,93–96. Relationship of MDSCs to other myeloid cells. Despite their morphological similarity, polymorphonuclear MDSCs and neutrophils are functionally and phenotypically different. First, polymorphonuclear MDSCs, but not neutrophils, are immunosuppressive97. Second, polymorphonuclear MDSCs express higher levels of CD115 and CD244 than neutrophils but lower levels of CXC‑chemokine receptor 1 (CXCR1) and CXCR2 (REFS 97,98). Finally, compared with neutrophils, polymorphonuclear MDSCs are less phagocytic, express higher levels of ARG1 and myeloperoxidase, have increased ROS production and show reduced chemotaxis towards supernatants from human carcinomas97,98. Similarly, although monocytic MDSCs and inflammatory monocytes share a common phenotype and morphology, these cell populations are functionally distinct. Monocytic MDSCs are highly immunosuppressive, as they express, among other factors, high levels of both iNOS and ARG1. By contrast, iNOS and ARG1 are not coordinately upregulated in monocytes. Furthermore, although in M1 macrophages iNOS expression is a hallmark of a tumoricidal phenotype, in monocytic MDSCs iNOS expression promotes suppressive activities36. This shift in iNOS activity probably reflects the interplay of iNOS with other enzymes expressed by MDSCs, such as ARG1 and NADPH oxidase, as the coordinated activity of these enzymes was shown to promote the production of peroxynitrite, which inhibits the proliferation, effector functions and migration of T cells99–102. Although differences exist in the expression of ARG1 and iNOS among mouse and human myeloid cells (for example, ARG1 is constitutively expressed by human granulocytes103 but not monocytes), evidence indicates that human MDSCs can also co-express these enzymes96,104. The MDSC population includes direct progenitors of DCs, macrophages and granulocytes. After 24 hours of in vitro culture, polymorphonuclear MDSCs phenotypically and functionally resemble neutrophils97. Culture of tumour-derived MDSCs in the absence of tumour-derived factors, or the transfer of MDSCs to tumour-free recipients, results in the generation of mature macrophages and DCs105–107. By contrast, the presence of tumour-derived soluble factors or adoptive transfer into tumour-bearing hosts promotes the differentiation of MDSCs into immuno suppressive macrophages107,108. MDSCs can also differentiate into DCs following transfer into tumour-bearing recipients109, but whether these DCs are immunosuppressive is not currently known. Furthermore, hypoxia in the tumour microenvironment drives the differentiation of MDSCs into TAMs109,110 (FIG. 3). Immunomodulatory functions of MDSCs. MDSCs exploit a plethora of redundant mechanisms to influence both innate and adaptive immune responses (FIG. 4). Broadly speaking, these mechanisms can be grouped into four classes. The first type of mechanism is the depletion of nutrients required by lymphocytes — specifically, l‑arginine depletion through ARG1‑dependent consumption49 and l‑cysteine deprivation via its consumption and sequestration111. The depletion of these amino acids causes downregulation of the ζ‑chain in the T cell receptor (TCR) complex and proliferative arrest of antigen-activated T cells. The second type of mechanism is the generation of oxidative stress, which is caused by the production of ROS and reactive nitrogen species by MDSCs. Peroxynitrite and hydrogen peroxide are produced by the combined and cooperative activities of NADPH oxidase, ARG1 and iNOS in different MDSC subsets, and these reactive species drive several molecular blocks in T cells, ranging from the loss of TCR ζ‑chain expression112 and interference with IL‑2 receptor signalling 113 to the nitration and subsequent desensitization of the TCR101. NATURE REVIEWS | IMMUNOLOGY VOLUME 12 | APRIL 2012 | 259 © 2012 Macmillan Publishers Limited. All rights reserved REVIEWS Natural TReg cells A subset of TReg cells that undergoes maturation in the thymus, where these cells acquire the ability to recognize with intermediate avidity self antigens presented by host MHC class II molecules before being released to the periphery. Induced TReg cells A subset of TReg cells that is derived from the direct conversion of CD4+ naive T cells in peripheral lymphoid organs in several situations, including the interaction with tumour-conditioned myelomonocytic cells in tumour-bearing hosts. TH17 cell (T helper 17 cell). A CD4+ T helper cell that produces IL‑17 and that is thought to be important in mediating host defence against certain infections, particularly at mucosal tissues. These cells are also thought to drive pathology in certain inflammatory and autoimmune diseases, such as Crohn’s disease. The third type of mechanism interferes with lymphocyte trafficking and viability. Expression of ADAM17 (disintegrin and metalloproteinase domaincontaining protein 17) at the plasma membrane of MDSCs decreases CD62L expression on the surface of naive CD4+ and CD8+ T cells, thereby limiting T cell recirculation to lymph nodes114. Another example is the modification of CCL2 by MDSC-derived peroxynitrite, a process that impairs the migration of effector CD8+ T cells to the tumour core102. Furthermore, MDSCs express galectin 9, which binds to T cell immunoglobulin and mucin domain-containing protein 3 (TIM3) on lymphocytes and induces T cell apoptosis115. MDSCs also decrease the number and inhibit the function of mouse and human NK cells, mostly through membrane contact-dependent mechanisms (that is, through membrane-bound TGFβ (in the case of mouse MDSCs) and through interaction with the NK cell receptor NKp30 (also known as NCR3))116–118. The fourth type of mechanism is the activation and expansion of TReg cell populations. MDSCs promote the clonal expansion of antigen-specific natural TReg cells and also induce the conversion of naive CD4+ T cells into induced TReg cells. The mechanisms are not completely understood, but may involve cell-to-cell contact (including CD40–CD40L interactions)119, the production of soluble factors (such as IFNγ, IL‑10 and TGFβ) by MDSCs120 and possibly also the expression of ARG1 by MDSCs121 (FIG. 4). Human CD14+HLA-DRlow/– MDSCs promote the transdifferentiation of TH17 cells into FOXP3+ induced TReg cells by producing TGFβ and retinoic acid122. In peripheral lymphoid organs, MDSC-mediated suppression of CD8+ T cells is usually antigen specific and requires the presentation of antigens by MDSCs and direct MDSC–T cell contact 101,123. By contrast, in the periphery 124 and at the tumour site109,110,125,126, the activity of MDSCs is enhanced by activated T cells, and as a result MDSCs are able to suppress nearby T cells in an antigen-nonspecific manner. However, if T cells are activated and begin to express FAS ligand (also known as CD95L), they may induce the apoptosis of FAS + MDSCs124. The co-dependence of cells in the myeloid lineage is further demonstrated by the regulation of mature DCs and macrophages by MDSCs. Through an IL‑10and cell contact-dependent mechanism, MDSCs skew macrophages towards an M2 phenotype by decreasing macrophage production of IL‑12 (REF. 127). The downregulation of IL‑12 is exacerbated by the macrophages themselves, as macrophages promote the production of IL‑10 by MDSCs (FIG. 4). As MDSC potency is increased by inflammation 128, it is not unexpected that inflammation enhances the crosstalk between MDSCs and macrophages. Inflammation mediates these effects by upregulating the expression of CD14 and increasing signalling through the TLR4 pathway in MDSCs129. MDSCs similarly impair DC function by producing IL‑10, which inhibits TLR-induced IL‑12 production by DCs and reduces DC‑mediated activation of T cells130. Mechanisms of tumour impact on myeloid cells Neoplastic cells and tumour-associated stromal cells release multiple tumour-derived soluble factors that perturb the myeloid compartment. Cytokines such as granulocyte–macrophage colony-stimulating factor (GM‑CSF), G‑CSF, M‑CSF, stem cell factor (SCF; also known as KIT ligand), VEGF and IL‑3 promote myelopoiesis and contribute, in part, to a blockade of myeloid cell maturation86,105 (FIG. 5). Tumour-derived soluble factors that are pro-inflammatory (such as IL‑1β, IL‑6, S100A8 and S100A9)131–133, as well as cytokines released by activated T cells (such as IFNγ, IL‑4, IL‑10 and IL‑13)125, initiate the immunosuppressive pathways that commit immature myeloid cells to become MDSCs and then further promote the differentiation of MDSCs towards immunosuppressive macrophages and DCs (FIG. 5). The tumour-derived factors CCL2, CCL12, CXC‑chemokine ligand 5 (CXCL5), prokineticin 2, S100A8 and S100A9 recruit immature myeloid cells to the tumour stroma68,134,135. Immature myeloid cells are also attracted by CCL2 that is nitrated or nitrosylated in the tumour environment. By contrast, effector CD8+ T cells are not recruited by modified CCL2, which may explain the selective enrichment of myelomonocytic cells within mouse and human tumours102. LPS, in combination with IFNγ, promotes the expansion of MDSC populations, probably by inhibiting DC differentiation136. Tumour-derived TGFβ also regulates MDSC accumulation137 and neutrophil polarization73 (FIG. 5). Neoplastic cells and their associated stromal cells also release into the bloodstream subcellular components known as exosomes, which contain signal peptides, mRNAs, microRNAs and lipids and promote MDSC population expansion (reviewed in REF. 138). Tumour-derived soluble factors regulate myeloid lineage cells on multiple levels through a variety of trans cription factors86,105,128 (FIG. 5), with signal transducer and activator of transcription 3 (STAT3) playing a major role. Early studies identified STAT3 as a crucial regulator of DC and macrophage defects139,140 and MDSC population expansion141–143. STAT3 not only prevents apoptosis and promotes cell proliferation by upregulating the antiapoptotic or pro-proliferative factors B cell lymphoma XL (BCL-XL), MYC, cyclin D1 and survivin105,144, but also regulates the expression of multiple proteins that are crucial for the differentiation of myeloid cells. One such pathway involves the calcium-binding pro-inflammatory proteins S100A8 and S100A9 (REF. 145). STAT3‑mediated upregulation of these proteins in myeloid progenitors inhibits DC differentiation and promotes MDSC accumulation146. S100A8 and S100A9 also enhance the suppressive activity of MDSCs and recruit MDSCs to the tumour site132. Myeloid cell NADPH oxidase is another important target of STAT3. STAT3‑mediated upregulation of the NADPH oxidase components p47phox (also known as NCF1) and gp91phox (also known as CYBB) increases ROS levels, thereby making MDSCs more suppressive90. STAT3 also downregulates protein kinase Cβ isoform II (PKCβII), which is required for DC differentiation, and thus prevents the development of DCs into mature cells147. In addition, STAT3 regulates the transcription factor 260 | APRIL 2012 | VOLUME 12 www.nature.com/reviews/immunol © 2012 Macmillan Publishers Limited. All rights reserved F O C U S O N t u m our i m m uno lo g y & i m m unotRh py E er V I EaW S a b T cell TReg cell IL-10 TGFβ ↓ L-arginine L-cystine ↑ FOXP3 CD40L ↓ L-arginine Altered mRNA transcription CAT2B • Loss of TCRζ chain • Proliferative arrest PI3K mTOR T cell L-arginine Cell cycle RAGE S100A8 S100A9 Loss of TCRζ chain H2O T cell ARG1 L-cysteine c H2O2 ARG1 ASC L-cystine MDSC HuR EIF2A ↓ L-cysteine MHC class II CD40 TCR GCN2 L-arginine Xc– TCR Chemotaxis S100A8 S100A9 Cyclin D3 mRNA instability Proliferative arrest T cell NOX iNOS O2– NO STAT3 STAT6 STAT1 C/EBPβ HIF1α MYD88 ↓ L-arginine d TGFβ NK cell ADAM17 CD62L Naive T cell IL-10 ONOO– Inhibition of NK cells CD8 GAL9 TIM3 Nitration or nitrosylation of TCR, CD8 and CD3 Nitrosylation ↑ cGMP PI3K–AKT STAT5 ERK2 Nitration or nitrosylation of CCL2 CD3 TCR CD8 Apoptotic T cell TCR CD8 CD3 CCR2 Apoptosis IL-2R T cell IL-2 Effector T cell ↑ IL-10 ↓ IL-12 T cell T cell Macrophage Decreased effector T cell activation Inhibition of T cell recruitment Figure 4 | Mechanisms of MDSC-dependent inhibition of T cell activation and proliferation. Myeloid-derived suppressor cells (MDSCs) can inhibit efficient antitumour T cell responses through a number ofNature mechanisms. Reviews | Immunology a | Tumour-associated MDSCs induce the development of regulatory T (TReg) cells or expand existing TReg cell populations. The calcium-binding proteins S100A8 and S100A9 are involved in the chemotaxis of MDSCs and other myeloid cells; these effects are mediated in part through the activation of receptor for advanced glycation end-products (RAGE). At the same time, S100A8 and S100A9 along with gp91phox are part of the NADPH oxidase (NOX) complex that is responsible for the increased production of reactive oxygen species (ROS) by MDSCs. b | Tumour-associated myeloid cells deprive T cells of amino acids that are essential for their growth and differentiation. c | Tumour-associated myeloid cells release oxidizing molecules, such as hydrogen peroxide (H2O2) and peroxynitrite (ONOO–). Peroxynitrite causes nitration and nitrosylation of components of the T cell receptor (TCR) signalling complex, and H2O2 causes the loss of the TCR ζ‑chain, thereby inhibiting T cell activation through the TCR. d | Tumour-associated myeloid cells can also interfere with T cell migration and viability. The metalloproteinase ADAM17 (disintegrin and metalloproteinase domain-containing protein 17) cleaves CD62L, which is necessary for T cell migration to draining lymph nodes, and galectin 9 (GAL9) can engage T cell immunoglobulin and mucin domain-containing protein 3 (TIM3) on T cells to induce apoptosis. As the induction of the immunosuppressive pathways that are depicted in the figure is regulated by common transcription factors, these pathways can operate in more than one myeloid cell type (see FIG. 5). ARG1, arginase 1; ASC, asc-type amino acid transporter; CAT2B, cationic amino acid transporter 2 isoform 1 (l-arginine transporter); CCL2, CC‑chemokine ligand 2; CCR2, CC‑chemokine receptor 2; C/EBPβ, CCAAT/enhancer-binding protein-β; EIF2A, eukaryotic translation initiation factor 2A; ERK2, extracellular signal-regulated kinase 2; FOXP3, forkhead box P3; HIF1α, hypoxia-inducible factor 1α; HuR, Hu-antigen R (also known as ELAVL1); IL, interleukin; IL‑2R, IL‑2 receptor; iNOS, inducible nitric oxide synthase; mTOR, mammalian target of rapamycin; MYD88, myeloid differentiation primary-response protein 88; NK, natural killer; PI3K, phosphoinositide 3‑kinase; STAT, signal transducer and activator of transcription; TGFβ, transforming growth factor‑β; Xc–, cystine–glutamate transporter. NATURE REVIEWS | IMMUNOLOGY VOLUME 12 | APRIL 2012 | 261 © 2012 Macmillan Publishers Limited. All rights reserved REVIEWS Tumour microenvironment Cytokine or immune mediator Transcription factor IL-1β, IFNγ IL-1β, IL-6, IL-10, IL-11, G-CSF, GM-CSF, VEGF, HSP70 GM-CSF IL-4, IL-13 TLR ligands TGFβ STAT1 STAT3 STAT5 STAT6 NF-κB SMADs S100A8, S100A9, NOX2, BCL-XL, cyclin D1, survivin, MYC, C/EBPβ, PKCβII BCL-XL, cyclins, survivin, MYC ARG1; NOX2 and antioxidants regulating ROS PGE2; NOX2 and antioxidants regulating ROS Targets of iNOS, ARG1 transcription Relevant myeloid cells MDSC MDSC TAM Suppressive DC TAM Immune suppression mediated by NO and ARG1 Population expansion; immune suppression mediated by ROS MDSC MDSC TAM Population expansion M2-type polarization; immune suppression Blockade of DC activation; immune suppression Immune M2-type suppression polarization; immune suppression MDSC TAM Neutrophil Neutrophil Mobilization Mobilization from bone from bone marrow; Polarization of immune neutrophils to suppression a pro-tumour phenotype Figure 5 | Molecular mechanisms affecting the myeloid lineage in cancer. The tumour microenvironment secretes many different cytokines and immune mediators that affect myeloid progenitors as well as mature myeloid | Immunology cells by regulating the activity of multiple transcription factors. These transcription factors,Nature in turn,Reviews regulate the synthesis of their protein targets, thereby affecting myeloid cell functions. ARG1, arginase 1; BCL-XL, B cell lymphoma XL; C/EBPβ, CCAAT/enhancer-binding protein-β; DC, dendritic cell; G-CSF, granulocyte colony-stimulating factor; GM-CSF, granulocyte–macrophage colony-stimulating factor; HSP70, heat-shock protein 70; IFNγ, interferon-γ; IL, interleukin; iNOS, inducible nitric oxide synthase; MDSC, myeloid-derived suppressor cell; NF-κB, nuclear factor‑κB; NO, nitric oxide; NOX2, NADPH oxidase 2; PGE2, prostaglandin E2; PKCβII, protein kinase Cβ isoform II; ROS, reactive oxygen species; STAT, signal transducer and activator of transcription; TAM, tumour-associated macrophage; TGFβ, transforming growth factor‑β; TLR, Toll-like receptor; VEGF, vascular endothelial growth factor. CCAAT/enhancer-binding protein-β (C/EBPβ). C/EBPβ regulates myelopoiesis in healthy individuals148 and has a crucial role in controlling the differentiation of myeloid progenitors to functional MDSCs131. STAT3 is responsible, at least in part, for inducing the expansion of MDSC populations via upregulation of C/EBPβ and also has an indirect role in myeloid cell mobilization, accumulation and survival149. The transcription factor STAT1 regulates subsets of myeloid cells through its effects on iNOS expression and is crucial for immune suppression by macro phages and MDSCs81,108,125. Other characteristics of MDSCs and macrophages (including upregulated expression of ARG1 (REFS 150–152) and increased TGFβ production121,153), and possibly the expansion of MDSC populations154, are controlled by STAT6. IL‑4‑induced polarization of TAMs activates STAT6, which binds to the promoter of the gene encoding the demethylase jumonji domain-containing protein 3 (JMJD3; also known as KDM6B). Activated JMJD3 demethylates histone H3 lysine 27 at specific genetic loci, and this increases the expression of ARG1, YM1 and FIZZ1, resulting in M2 polarization155. However, the genetic ablation of Jmjd3 in mice causes a defect in interferonregulatory factor 4 (IRF4)-dependent M2 polarization in response to M‑CSF, helminth infection or chitin administration, but not following IL‑4 stimulation, suggesting a more complex regulatory network156. The TLR family also has a prominent role in myeloid cell development, primarily via the activation of myeloid differentiation primary-response protein 88 (MYD88) and the downstream induction of nuclear factor‑κB (NF‑κB). NF‑κB signalling is important for the mobilization of myeloid cells to sites of infection, injury or tumour growth157,158. TLR4 regulates the inflammation-driven suppressive potency of MDSCs through an NF‑κB-dependent mechanism129. The pro-inflammatory mediators COX2 and PGE2, which enhance MDSC accumulation and suppressive activity 137,159–161, are also potential targets for NF‑κB162. 262 | APRIL 2012 | VOLUME 12 www.nature.com/reviews/immunol © 2012 Macmillan Publishers Limited. All rights reserved F O C U S O N t u m our i m m uno lo g y & i m m unotRh py E er V I EaW S Autochthonous tumour Differently from transplanted tumours, which arise from the experimental transfer of neoplastic cells or tissues, autochthonous tumours develop spontaneously in the host. Autochthonous tumours can be derived from either chemical carcinogenesis or targeted tissue expression of oncogenes by genetic manipulation of mice. Model of MDSC involvement in tumour progression Recent studies of autochthonous tumour formation in transgenic mice indicate that cells with an MDSC phenotype probably intervene in the very early stages of cancer progression. Mice with autochthonous pancreatic cancer undergo progressive waves of myeloid cell recruitment after the initiation of the transforming programme driven by the Kras oncogene163. Recruited myeloid cells contribute to the local production of IL‑6 and IL‑11, which activate STAT3. STAT3, in turn, induces antiapoptotic and pro-proliferative genes, fuelling tumour initiation, promotion and progression164,165. During the early events of colitis-associated cancer, myeloid cells act as tumour promoters by enhancing the proliferation of tumour-initiating cells and by protecting premalignant intestinal epithelial cells from apoptosis166. In thyroid carcinomas, the oncogenic fusion protein RET/PTC3 directly induces the production of CCL2 and GM‑CSF, which together promote the recruitment of CD11b+GR1+ myeloid cells that can promote tumour progression167,168. An important question is whether these early recruited cells are immunosuppressive MDSCs. Unfortunately, only a few studies in autochthonous tumour models have determined the immunosuppressive activity of the tumourassociated CD11b+GR1+ cells. In models of spontaneous breast, pancreatic or lung cancer, accumulated myeloid cells had both the phenotype and immunosuppressive features of MDSCs109,163,169. Moreover, in a recent study, conditional deletion of the gene encoding p120 catenin (a protein involved in cell–cell adhesion and signal transduction) in mice caused the formation of invasive squamous cell cancer and desmoplasia and the associated production of CCL2, GM‑CSF, M‑CSF and TNF. These events resulted in the accumulation of immunosuppressive CD11b+GR1+CD124+ MDSCs that promoted tumour progression by activating stromal fibroblasts170. In another model of multistep squamous carcino genesis driven by the human papillomavirus 16 (HPV‑16) early-region genes (including the E6 and E7 oncogenes) under the control of the human keratin 14 promoter and enhancer, CD11b+GR1+F4/80–CD11c– MDSCs were the most abundant leukocyte subtype in premalignant skin, and they also accumulated progressively in the spleen. However, these cells failed to inhibit the polyclonal activation of either CD4+ or CD8+ T cells and did not produce ROS60. These results, together with the data discussed above from transplantable tumour models, support a two-stage model of MDSC involvement in cancer. An almost universal feature of tumour progression is the activation of abnormal myelopoiesis and the recruitment of immature myeloid cells into tissues. This process is governed by diverse soluble factors and is dependent on the upregulation of STAT3 and other key transcription factors (FIG. 5). Myelopoiesis during acute infection, stress or trauma results in rapid terminal differentiation of myeloid cells. By contrast, cancer myelopoiesis is associated with defective myeloid cell differentiation, which results in the accumulation and persistence of immature myeloid cells. Although necessary, these events are not sufficient to generate immunosuppressive MDSCs: activation of the immature myeloid cells via a network of regulatory mechanisms is also required (FIG. 5). The activation of these mechanisms in mice with most transplantable tumours and many, but not all, spontaneous tumours results in the accumulation of immunosuppressive MDSCs. In tumour sites, these cells further differentiate into TAMs and possibly into suppressive DCs. In patients with cancer, cells with an MDSC phenotype are almost universally immuno suppressive, which may reflect their isolation from patients with advanced disease. If immunosuppressive activity is not a property of the first wave of immature myeloid cells recruited to tumours, continuous stimulation of myelopoiesis and activation of immature myeloid cells by tumour-derived soluble factors may drive the subsequent accumulation of immunosuppressive MDSCs that support tumour promotion and form the metastatic niche. Accordingly, the oncogenic programme may have a greater influence on the functional immunosuppressive activities of MDSCs than on their accumulation. The transition from immature myeloid cell to MDSC might be defective in some experimental tumour models, and different oncogenic programmes may differentially affect the kinetics of immature myeloid cell to MDSC conversion. Treatment in vitro with combinations of GM‑CSF, G‑CSF, IL‑6 and IL‑13 induces the rapid differentiation of human and mouse bone marrow precursor cells into cells that resemble MDSCs89,131,137,171. These studies may provide the framework for identifying the key molecules that govern MDSC maturation. Therapeutic targeting of myeloid cells It is increasingly clear that successful cancer immunotherapy will require limiting the immunosuppressive effects of populations of myeloid cells. Knowledge of the molecular mechanisms responsible for the accumulation of MDSCs and immunosuppressive macrophages and DCs in cancer has allowed for therapeutic targeting of these cells. This targeting is focused on six main goals. The first goal is to inhibit the molecular mechanisms used by myeloid cells to block lymphocyte reactivity and proliferation. Second, targeting should inhibit the generation of MDSCs from bone marrow progenitors or induce the apoptosis of circulating MDSCs. Third, it should force MDSCs to mature into proficient APCs that can stimulate tumour-specific T cells. Fourth, it should prevent the trafficking of myeloid cells from the bone marrow to peripheral lymphoid organs and to tumours. The fifth goal is to repolarize or eliminate TAMs and replace them with pro-inflammatory M1 macrophages that enhance antitumour immune responses. Finally, the sixth goal is to restore the antigen-presenting capabilities of DCs and macrophages, allowing these populations to activate effector T cell responses (TABLE 2). The proposed two-stage model for MDSC involvement might have implications for the further development of therapies. Some immunosuppressive mechanisms are common to all myeloid cells, but others are unique to individual populations. Therefore, targeting common effector molecules is likely to be more effective than targeting individual suppressive pathways. NATURE REVIEWS | IMMUNOLOGY VOLUME 12 | APRIL 2012 | 263 © 2012 Macmillan Publishers Limited. All rights reserved REVIEWS Table 2 | Pharmacological regulation of myeloid cells in cancer Therapeutic treatment Type of cancer* Molecular events Effects on myeloid cells Refs Nitroaspirin Colon carcinoma Downregulation of ARG1, iNOS and peroxynitrite in MDSCs Inhibition of MDSC suppressive effects 172 Phosphodiesterase‑5 inhibitors (sildenafil and tadalafil) Mammary carcinoma, colon carcinoma and fibrosarcoma Downregulation of ARG1, iNOS, and CD124 (IL-4Rα) in MDSCs Inhibition of MDSC suppressive effects 96 AT38 (an NO donor based on the furoxan molecule) Fibrosarcoma and thymoma Downregulation of ARG1, iNOS and peroxynitrite in MDSCs; expression of nitrated or nitrosylated CCL2 Inhibition of MDSC suppressive effects; increased CD8+ T cell/MDSC ratio in tumours 102 Triterpenoids Colon carcinoma, lung carcinoma and thymoma Inhibition of ROS production by MDSCs Inhibition of MDSC suppressive effects 173 Tyrosine kinase inhibitor (sunitinib) Fibrosarcoma and colon, breast, lung and kidney cancer; human renal cell carcinoma Possible KIT blockade; STAT3 inhibition; GM‑CSF confers resistance by activating STAT5 in intratumoural MDSCs Inhibition of MDSC expansion in lymphoid organs but not in the tumour stroma; modest inhibition of MDSC population expansion in patients 174–178 Cyclooxygenase 2 inhibitors (SC58236, SC58125 and celecoxib) Mammary carcinoma, mesothelioma, lung carcinoma and glioma Downregulation of PGE2, ARG1, ROS and CCL2 and increase in the expression of CXCL10 by MDSCs Inhibition of MDSC suppressive effects 137,162, 179 KIT-specific antibody Colon carcinoma Blockade of the KIT–SCF interaction Inhibition of MDSC population expansion 180 CSF1R and KIT receptor tyrosine kinase inhibitor (PLX3397) Mammary carcinoma Blockade of CSF1R and KIT 181 CCL2-specific monoclonal antibody Mammary carcinoma Interference with CCL2 binding to Inhibition of metastatic spread by CCR2 and with VEGFA upregulation targeting inflammatory monocytes and macrophages Amino-bisphosphonate (zoledronate) Mammary tumours and mesothelioma Reduction in VEGF and pro-MMP9 serum levels Inhibition of MDSC population expansion Very small size proteoliposomes Lymphomas and sarcoma iNOS downregulation Changes in MDSC subset distribution 184 Antagonists of CXCR2 (S‑265610) and CXCR4 (AMD3100) Breast cancer Interference with the chemokines CXCL12 and CXCL5 Altered recruitment of immature myeloid cells to the tumour 134 Prokineticin 2-specific antibody Various human and mouse tumours in nude mice Interference with prokineticin 2 pleiotropic activity Inhibition of polymorphonuclear MDSC population expansion and recruitment to tumour and pre-metastatic niches 68,69 CSF1R antagonist (GW2580) Lung carcinoma and prostate cancer CSF1R interference; downregulation of ARG1 in MDSCs; reduction in VEGF and MMP9 levels in the tumour Inhibition of the expansion of MDSC and macrophage populations and their recruitment to the tumour 185 VEGF-Trap (aflibercept), VEGF-specific antibody (bevacizumab (Avastin; Genentech/Roche)) Various human solid tumours and human metastatic renal cell cancer VEGF interference blocks tumour growth Increased functional maturation of DCs 186,187 Gemcitabine Lung cancer and breast cancer MDSC apoptosis Inhibition of MDSC population expansion 127,188 5-fluorouracil Thymoma MDSC apoptosis Inhibition of MDSC population expansion 189 Doxorubicin– cyclophosphamide Human breast cancer May induce MDSC apoptosis Weak inhibition of MDSC population expansion Docetaxel Mammary carcinoma MDSC apoptosis; differentiation of surviving cells to M1 macrophages Inhibition of MDSC population expansion; macrophage polarization to M1 phenotype All-trans retinoic acid Sarcoma and colon carcinioma; human metastatic renal cell carcinoma Differentiation of immature myeloid Inhibition of MDSC accumulation cells to mature leukocytes Vitamin D3 Human head and neck cancer Forced differentiation of CD34+ immature myeloid cells Moderate inhibition of MDSC population expansion Combined treatment with IL‑12, CCL16, CpG DNA and an IL‑10 receptor-specific monoclonal antibody Lung cancer and breast cancer Decreased levels of IL‑10, CCL2 and TGFβ; increased levels of TNF, IL‑15 and IL‑18. TAM reprogramming Inhibition of TAM recruitment 264 | APRIL 2012 | VOLUME 12 50 182,183 88 190 191,192 193 194,195 www.nature.com/reviews/immunol © 2012 Macmillan Publishers Limited. All rights reserved F O C U S O N t u m our i m m uno lo g y & i m m unotRh py E er V I EaW S Table 2 (cont.) | Pharmacological regulation of myeloid cells in cancer Therapeutic treatment Type of cancer* Molecular events Effects on myeloid cells Tumour-specific CTLs engineered to release IL‑12; chimeric antigen receptor-expressing T cells engineered to release IL‑12 Melanoma and colon carcinoma Increased antigen cross-presentation and co-stimulation; acute inflammation signature (including IFNγ production); increased TNF production DC, MDSC and TAM reprogramming CD40-specific monoclonal antibodies plus IL‑2 Renal cell carcinoma Increased iNOS and TIMP1 expression TAM reprogramming in lung metastases but not in the primary tumour 198 Agonist CD40-specific monoclonal antibodies and gemcitabine Pancreatic carcinoma; human pancreatic ductal adenocarcinoma Targeting and activation of blood circulating macrophages TAM reprogramming 199 Downregulation of placental growth factor TAM reprogramming 200 IL‑12 production by NK cells; TAMs become IL‑12hiIL‑10lowMHC-IIhiARG1low TAM reprogramming to M1 macrophage phenotype 201 Histidine-rich glycoprotein Fibrosarcoma, pancreatic (HRG) cancer and breast cancer Inhibition of NF‑κB signalling by targeting IκB kinase Ovarian cancer Refs 196,197 ARG1, arginase 1; CCL, CC‑chemokine ligand; CCR2, CC‑chemokine receptor 2; CSF1R, macrophage colony-stimulating factor receptor; CTL, cytotoxic T lymphocyte; CXCL, CXC-chemokine ligand; DC, dendritic cell; GM‑CSF, granulocyte–macrophage colony-stimulating factor; IFNγ, interferon‑γ; IκB, NF‑κB inhibitor; IL, interleukin; iNOS, inducible nitric oxide synthase; MDSC, myeloid-derived suppressor cell; MMP, matrix metalloproteinase; NF‑κB, nuclear factor-κB; NK, natural killer; NO, nitric oxide; PGE2, prostaglandin E2; ROS, reactive oxygen species; SCF, stem cell factor; STAT, signal transducer and activator of transcription; TAM, tumour-associated macrophage; TGFβ, transforming growth factor-β; TIMP1, tissue inhibitor of metalloproteinases 1; TNF, tumour necrosis factor; VEGF, vascular endothelial growth factor. *Human cancers are indicated; other studies were conducted in mouse models. 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