Abstract

Introduction

Myeloid cells are a highly diverse population. Mononuclear myeloid cells include terminally differentiated macrophages and dendritic cells (DC), as well as monocytes, which under inflammatory conditions differentiate in tissues to macrophages and DCs. Granulocytic myeloid cells include populations of terminally differentiated polymorphonuclear neutrophils, eosinophils, basophils, and mast cells. Myelopoiesis in response to pathogenic stimuli is a fundamental mechanism protecting the host. It largely manifests in expansion of activated neutrophils and monocytes. Classical activation of these cells takes place in a response to strong signals that usually come in form of pathogen-associated molecular patterns (PAMPs) or danger-associated molecular patterns (DAMPs) molecules. This activation is relatively short-lived and manifests in robust phagocytosis, respiratory burst, and release of pro-inflammatory cytokines. It terminates upon cessation of the stimuli. In contrast, persistent stimulation associated with chronic infection, inflammation, or cancer involves relatively low-strength signals. This induces modest but persistent myelopoiesis. Myeloid cells generated under these conditions, although similar to neutrophils and monocytes in morphology and phenotype, have different genomic and biochemical profiles and functional activity. The main functional characteristic of these cells is their potent ability to suppress various types of immune responses. It is possible that this mechanism evolved as a form of protection from extensive tissue damage caused by an uncontrolled immune response associated with unresolved inflammation.

Reports on the accumulation of immune suppressive myeloid cells associated with tumor progression were published sporadically beginning in the early 1970s (1). During the 1980s and early 1990s, work from the laboratories of Diana Lopez, Jim Talmadge, M. Rita Young, and Hans Schreiber, demonstrated that various types of myeloid cells could inhibit immune function in cancer. However, the specific nature and biological significance of these cells remained largely unclear. The field started changing in the late 1990s when the Gr1⁺CD11b⁺ cellular phenotype was suggested as defining the immune suppressive myeloid cells in spleens of mice and when these cells were shown to be phenotypically similar but functionally distinct from monocytes and neutrophils (2, 3). The observations of accumulation of large numbers of these cells in spleens and tumors with potent immune suppressive activity were readily reproducible in most murine tumor models. However, it quickly became apparent that CD11b⁺Gr-1⁺ cells were heterogeneous. Different phenotypic criteria and multiple mechanisms of action were used to define these cells. In 2007, in an attempt to unify different descriptions of these cells, the name myeloid-derived suppressor cells (MDSC) was proposed (4). This name was based on the myeloid origin of the cells and their main functional trait – potent immune suppressive activity. In the following years, interest in these cells skyrocketed with almost 2,500 papers published in less than 10 years. MDSC were implicated in various aspects of immune regulation, not only cancer, but also in diseases that involve chronic inflammation, infection, autoimmune diseases, trauma, graft versus host disease, etc. Evidence of the clinical significance of MDSC in cancer has emerged, and MDSC have become an important part of the tumor immunology field. However, as often happens with most teenagers, MDSC periodically have an identity crisis and a difficult relationship with the more established cells in the field. Only recently have MDSC entered a more mature age where their identity and place among other myeloid cells has become clear.

Main phenotypic and functional characteristics of MDSC

MDSC consist of two large groups of cells termed granulocytic or polymorphonuclear (PMN-MDSC), which are phenotypically and morphologically similar to neutrophils; and monocytic (M-MDSC) – phenotypically and morphologically similar to monocytes. Therefore phenotypic criteria alone are not sufficient to identify cells as MDSC. In most types of cancer, PMN-MDSC represent more than 80% of all MDSC. In addition to these two main populations, MDSC include a small group (less than 3%) of cells with myeloid colony forming activity representing a mixture of myeloid progenitors and precursors. In mice, MDSC were mostly described in bone marrow, peripheral blood, spleen, liver, lung, or tumors of various organs. PMN-MDSC can be defined as CD11b⁺Ly6G⁺Ly6C^lo and M-MDSC as CD11b⁺Ly6G⁻Ly6C^hi, with other markers under investigation. In humans, MDSC were mostly described in blood and tumors of various organs with number of studies describing these cells in bone marrow. Criteria for the phenotypic characterization of these cells by flow cytometry are now relatively well defined (5, 6). Among peripheral blood mononuclear cells (PBMC), PMN-MDSC are defined as CD11b⁺CD14⁻CD15⁺ or CD11b⁺CD14⁻CD66b⁺, and M-MDSC as CD11b⁺CD14⁺HLA-DR^−/loCD15⁻. Lin⁻ (including CD3, CD14, CD15, CD19, CD56) HLA-DR⁻CD33⁺ cells contain mixed groups of MDSC comprising more immature progenitors. The term “early-stage MDSC” (e-MDSC) was recently proposed for this latter population (7).

In humans, M-MDSC could be separated from monocytes based on the expression of MHC class II molecule - HLA-DR. Until recently, the only method allowing for separation of neutrophils from PMN-MDSC in humans was gradient centrifugation using a standard Ficoll gradient. PMN-MDSC are enriched in the low-density fraction (PBMC), whereas neutrophils are high density cells (8). Recently, we identified lectin-type oxidized LDL receptor 1 (LOX-1) as a marker of PMN-MDSC in humans (9). If confirmed in further studies, LOX-1 expression on neutrophils can be used for direct identification of PMN-MDSC in blood and tissues. In mice, the phenotypic distinction between neutrophils and PMN-MDSC in the same mouse is difficult. Several different markers were suggested but thus far, none of them allow for definitive identification of PMN-MDSC.

Immune suppression is a main feature of MDSC. Although MDSC were implicated in suppression of different cells of the immune system, the main targets of MDSC are T cells. The main factors implicated in MDSC-mediated immune suppression include arginase (ARG1), iNOS, TGFβ, IL-10, COX2, indoleamine 2,3-dioxygenase (IDO) sequestration of cysteine, decrease of L-selectin expression by T-cells and many others. In recent years, it became clear that M-MDSC and PMN-MDSC utilize different mechanisms of immune suppression. M-MDSC suppress T-cell responses both in antigen-specific and non-specific manners utilizing mechanisms associated with production of NO and cytokines (reviewed in (10)). PMN-MDSC, on the other hand, are capable of suppressing immune responses primarily in an antigen-specific manner. Induction of antigen-specific T-cell tolerance is one of the major characteristics of these cells (11, 12). Reactive oxygen species (ROS) production is essential for this ability. Reaction of NO with superoxide generates peroxynitrite (PNT), which directly inhibits T-cells by nitrating T-cell receptors and reducing their responsiveness to cognate antigen-MHC complexes (13). PNT also reduces the binding of antigenic peptides to MHC molecules on tumor cells (14) and blocks T-cell migration by nitrating T-cell specific chemokines (15). (Fig. 1)

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Figure

Development and function of MDSC

Tumor derived factors affect different stages of myeloid cell differentiation resulting in generation of pathologically activated M-MDSC and PMN-MDSC. HSC-hematopoietic stem cells; CMP – common myeloid progenitor; GMP-granulocyte-macrophage progenitor; MDP – macrophage/dendritic cell progenitors. PMN-MDSC and M-MDSC migrate to lymphoid organs and to tumor site. The function and fate of these cells is different in different sites. In peripheral lymphoid organs PMN-MDSC retain high level of various ROS and cause antigen-specific T-cell suppression/tolerance. M-MDSC produce large array of different factors that enable these cells suppress not only antigen-specific but also non-specific T cell responses. M-MDSC maintain high activity of STAT3 that prevent their quick differentiation to DCs or macrophages. In tumor site, largely due to the effect of hypoxia STATA3 activity in MDSC is dramatically reduced. This result in rapid differentiation of M-MDSC to tumor associated macrophages (TAM). ROS level in PMN-MDSC is substantially reduced, but up-regulation of arginase 1(ARG1) and other factors responsible for nonspecific T-cell suppression is increased. The same happens with M-MDSC. PMN-MDSC are dying rapidly. Factors released by dying cells can contribute to immune suppressive mechanisms.

The large number of different immune suppressive mechanisms described for MDSC does not mean that these mechanisms are simultaneously operational. The prevalence of a particular immune suppressive mechanism depends on the type of MDSC expanded, as well as on the stage of the disease and the site where the suppression is occurring. It is likely that at any given time there is a dominant suppressive mechanism used by MDSC and that this mechanism could change throughout the progression of the disease.

Besides immune suppressive mechanisms, MDSC promote tumor progression by affecting the remodeling of the tumor microenvironment and tumor angiogenesis via production of VEGF, bFGF, Bv8, and MMP9 (16–18). MDSC were implicated in the formation of pre-metastatic niches (19–22), and the promotion of metastases by infiltrating primary tumors (23, 24). CD11b⁺Gr1⁺ cells were shown to oppose cellular senescence in a model of spontaneous prostate cancer by antagonizing IL-1α mediated senescence (25). In contrast, a recent report indicated that CCR2⁺ myeloid cells, represented largely by monocytic cells, supported senescence in a model of liver cancer (26).

Major mechanisms regulating MDSC accumulation and differentiation

Accumulation of MDSC is a complex phenomenon. We have previously proposed a two-signal model describing this process (27). This model asserts that accumulation of MDSC requires two distinct although partially overlapping types of signals: the first is responsible for the expansion of immature myeloid cells associated with inhibition of their terminal differentiation, and the second is responsible for the pathological activation of these cells, converting immature myeloid cells to MDSC.

The first group of signals is mostly driven by tumor-derived growth factors and involves such factors as STAT3, IRF8, C/EBPβ, Notch, adenosine receptors A2b signaling, NLRP3 (reviewed in (27)). Recently, the retinoblastoma protein 1 (Rb1) was implicated in the ability of some M-MDSC to differentiate to PMN-MDSC. While Rb1^hi M-MDSC mainly gave rise to macrophages and DC, the vast majority of Rb1^lo M-MDSC differentiated towards PMN-MDSC (28). Recently, the accumulation of Rb1^lo Ly6G⁺ PMN-MDSC was confirmed in the PyMT transgenic model of breast cancer (29).

The second group of signals are mediated by factors produced mostly by the tumor stroma (pro-inflammatory cytokines, HMGB1) and include the NF-κB pathway, STAT1, STAT6, prostaglandin E2 (PGE2) and cyclooxygenase 2 (COX2) (reviewed in (27)). Recently, the endoplasmic reticulum (ER) stress response pathway was implicated in the suppressive activity of MDSC. The ER stress response is an evolutionary conserved mechanism developed to protect cells from various stress conditions including hypoxia, nutrient deprivation, low pH, etc. MDSC isolated from tumor-bearing mice and cancer patients overexpressed several markers of ER stress including sXBP1 and CHOP, and displayed an enlarged endoplasmic reticulum, one of the hallmarks of ER stress (30). Administration of an ER stress inducer to tumor-bearing mice was shown to increase the accumulation of MDSC and their suppressive activity (31). Induction of ER stress with thapsigargin converted human neutrophils to immune suppressive PMN-MDSC (9). The transcription factor CHOP was implicated in the suppressive activity of MDSC in the tumor site. CHOP-deficient MDSC lost the ability to suppress T-cells stimulated in an antigen non-specific manner and were even able to stimulate T-cells (32). However, CHOP-deficient MDSC retained the ability to suppress the T-cell response in an antigen-specific stimulation (33). More studies will be necessary to clarify the role of the specific mechanisms of ER stress responses in MDSC function.

In tumor tissues, M-MDSC rapidly differentiate to TAM (34, 35). A recent study implicates down-regulation of STAT3 activity in tumor associated M-MDSC in this phenomenon. This effect was controlled by hypoxia-inducible activation of CD45 phosphatase in these cells.

MDSC place among other myeloid cells

MDSC are pathologically activated myeloid cells. This raises the question of whether MDSC are really different from neutrophils and monocytes. In many studies, cells with typical MDSC features were called monocytes and neutrophils, and cells were called MDSC even in the absence of those features. Accumulated data in recent years allow us to make a conclusion about the specific nature of MDSC. This conclusion is based on several lines of evidence.

• Immune suppressive activity is intrinsic feature of MDSC. Mature neutrophils or monocytes cannot be converted to potent immune suppressive cells in vitro (at a level similar to MDSC) by simply activating them with PAMPs and DAMPs or pro-inflammatory cytokines. Moreover, in some cases neutrophils can promote antitumor response (36).

• Human PMN-MDSC have a unique genomic profile distinguishing them from neutrophils in the same patient, whereas neutrophils from healthy donors and cancer patients have very similar gene expression (9). Mouse MDSC were also characterized by specific proteome (37–39) and transcriptome (40, 41) profiles.

• Phenotypically, M-MDSC can be distinguished from TAMs by increased relative expression of F4/80, low to intermediate expression of Ly6C and low or undetectable expression of S100A9 protein, low expression of IRF8, and increased M-CSF receptor, CD115 (42). Most of the published data indicate that cells with the phenotype of inflammatory monocytes (CD11b⁺Ly6C^hiLy6G⁻) in tumors have potent immune suppressive activity and thus can be attributed to M-MDSC (43).

• There are number of biochemical features that clearly differentiate MDSC from their control counterparts. These features include high arginase and iNOS expression and activity, high and persistent level of ROS including superoxide, myeloperoxidase, hydroxyl peroxide, and peroxynitrite. PMN-MDSC and M-MDSC also can be distinguished from neutrophils and monocytes by their elevated ER stress response. More details are provided in a recent review (7).

Thus, MDSC represent a relatively stable, distinct state of functional activity of neutrophils and monocytes. Although PMN-MDSC concept is widely accepted, there are number of studies that use different approach to nomenclature of tumor associated neutrophils (TAN) based on concept of phenotypic plasticity of TAN, which is modulated through distinct micro-environmental signals, at different stages of tumor progression (36, 44). In these reports neutrophils with immunosuppressive and tumor promoting functions are called N2, as opposed to antitumor N1, neutrophils (8, 9). Although this point of view is definitely has some rationale, it is difficult to envision that short-lived, terminally differentiated PMN could be effectively polarized in tumor tissues. It is more likely that N1 cells represent activated bona fide PMN cells, whereas N2 cells are in fact PMN-MDSCs. Indeed, number of reports demonstrated potent immune suppressive activity by TAN, which defines these cells as PMN-MDSC (45–47).

Better characterization of MDSC in recent years shed new light on MDSC biology. Recent evidence has linked the accumulation of immature myeloid cells with an MDSC-like phenotype during chronic inflammation to the early stages of tumor development. Exposure of mice to cigarette smoke caused accumulation of these cells in lungs and spleens (48). However, only after the development of lung cancer did these cells become immune suppressive MDSC. Nevertheless, their depletion increased survival (48). In a model of skin carcinogenesis, accumulation of immature myeloid cells without suppressive function in the skin of mice promoted tumor development (49). This suggests that cells with an MDSC-like phenotype may play a significant role in tumor development and progression via mechanisms not necessarily related to their ability to suppress tumor-specific immune responses. It is possible that accumulation of MDSC is a gradual process. Myeloid progenitors and precursors affected by low-strength pathological signals coming from the developing tumor gradually acquire changes leading to their pathological activation. Bona fide MDSC is the latest stage of this process. Cells at intermediate stages (MDSC-like cells), though they do not possess potent immune suppressive activity, may actively contribute to tumor progression and metastases. Future study will determine whether this concept is correct.

Basic strategies to therapeutically target MDSC

Ample evidence supports a close association between MDSC accumulation and clinical outcome in cancer patients (50, 51). Recent meta-analysis of the studies of 442 patients with various solid tumors demonstrated that MDSC were highly significantly associated with poor overall and progression-free survival (52). MDSC have been implicated in resistance to anti-cancer therapies including sunitinib (53), cisplatin and other chemotherapeutics in lung cancer (54–56), and doxorubicin and melphalan in multiple myeloma (57). Recent studies show an association of MDSC level with patient response to CTLA4/ipilimumab (58, 59) and PD-1 (60, 61).

The fact that MDSC play an important role in the regulation of tumor growth has stimulated the search for a way to therapeutically target these cells.

MDSC can be eliminated with relatively low doses of chemotherapy with gemcitabine and 5-fluorouracil (62–64). It was recently shown that targeting the TRAIL receptor could be a potent and selective method of MDSC depletion (30). Peptibodies consisting of S100A9-derived peptides conjugated to antibody Fc fragments have shown potential in eliminating MDSC in mouse models (65).

MDSC can be functionally inactivated by targeting their suppressive machinery. Recent clinical reports indicated that head and neck and multiple myeloma cancer patients treated with the PDE-5 inhibitor tadalafil had fewer circulating MDSC, lower iNOS and arginase expression in these cells, and a greater number of spontaneously generated tumor specific T-cells (66–68). Nrf2 is a transcription factor that plays an important role in the cellular protection against free radical damage. Synthetic triterpenoid reduced the production of ROS by MDSC and their suppressive activity by upregulating Nrf2 (69). Inhibition of COX-2 downregulated the production of immune suppressive prostaglandin E2, and nitroaspirin has been shown to downregulate NO production (70, 71). Class I HDAC inhibitor entinostat was shown to have an inhibitory effect on MDSC (72), although the mechanism behind effect remains unclear.

MDSC expansion and differentiation can be targeted by all-trans-retinoic acid (ATRA) (73). In lung cancer patients, immune responses to a p53 vaccine were improved if the patients received a short course of ATRA (74). STAT3 inhibition can induce MDSC differentiation into immunogenic DC (75, 76). Phospholipid phosphatidylserine (PS) targeting antibody was shown to decrease frequency of MDSC in tumor-bearing mice although mechanism by which this occurs is unclear (77).

Conclusions

MDSC are a critical factor regulating immune responses under many pathological conditions and have recently became a prominent fixture of tumor immunology. However, their biological role can be established only if methods to selectively target these cells are developed. This requires specific markers of these cells to be identified, which would be possible if the molecular mechanisms governing the development of these cells were better characterized. Hopefully, the next couple of years will bring new and exciting data addressing those challenges.

Acknowledgments

Footnotes

References