The adaptor protein TRAF3 is an immune checkpoint that inhibits myeloid-derived suppressor cell expansion.

Myeloid-derived suppressor cells (MDSCs) are aberrantly expanded in cancer patients and under other pathological conditions. These cells orchestrate the immunosuppressive and inflammatory network to facilitate cancer metastasis and mediate patient resistance to therapies, and thus are recognized as a prime therapeutic target of human cancers. Here we report the identification of the adaptor protein TRAF3 as a novel immune checkpoint that critically restrains MDSC expansion. We found that myeloid cell-specific Traf3-deficient (M-Traf3 -/-) mice exhibited MDSC hyperexpansion during chronic inflammation. Interestingly, MDSC hyperexpansion in M-Traf3 -/- mice led to accelerated growth and metastasis of transplanted tumors associated with an altered phenotype of T cells and NK cells. Using mixed bone marrow chimeras, we demonstrated that TRAF3 inhibited MDSC expansion via both cell-intrinsic and cell-extrinsic mechanisms. Furthermore, we elucidated a GM-CSF-STAT3-TRAF3-PTP1B signaling axis in MDSCs and a novel TLR4-TRAF3-CCL22-CCR4-G-CSF axis acting in inflammatory macrophages and monocytes that coordinately control MDSC expansion during chronic inflammation. Taken together, our findings provide novel insights into the complex regulatory mechanisms of MDSC expansion and open up unique perspectives for the design of new therapeutic strategies that aim to target MDSCs in cancer patients.

Here, There, and Everywhere: Myeloid-Derived Suppressor Cells in Immunology.

Myeloid-derived suppressor cells (MDSCs) were initially identified in humans and mice with cancer where they profoundly suppress T cell- and NK cell-mediated antitumor immunity. Inflammation is a central feature of many pathologies and normal physiological conditions and is the dominant driving force for the accumulation and function of MDSCs. Therefore, MDSCs are present in conditions where inflammation is present. Although MDSCs are detrimental in cancer and conditions where cellular immunity is desirable, they are beneficial in settings where cellular immunity is hyperactive. Because MDSCs can be generated ex vivo, they are being exploited as therapeutic agents to reduce damaging cellular immunity. In this review, we discuss the detrimental and beneficial roles of MDSCs in disease settings such as bacterial, viral, and parasitic infections, sepsis, obesity, trauma, stress, autoimmunity, transplantation and graft-versus-host disease, and normal physiological settings, including pregnancy and neonates as well as aging. The impact of MDSCs on vaccination is also discussed.

The Receptor for Advanced Glycation Endproducts (RAGE) and Its Ligands S100A8/A9 and High Mobility Group Box Protein 1 (HMGB1) Are Key Regulators of Myeloid-Derived Suppressor Cells.

Immunotherapies including checkpoint blockade immunotherapy (CBI) and chimeric antigen receptor T cells (CAR-T) have revolutionized cancer treatment for patients with certain cancers. However, these treatments are not effective for all cancers, and even for those cancers that do respond, not all patients benefit. Most cancer patients have elevated levels of myeloid-derived suppressor cells (MDSCs) that are potent inhibitors of antitumor immunity, and clinical and animal studies have demonstrated that neutralization of MDSCs may restore immune reactivity and enhance CBI and CAR-T immunotherapies. MDSCs are homeostatically regulated in that elimination of mature circulating and intratumoral MDSCs results in increased production of MDSCs from bone marrow progenitor cells. Therefore, targeting MDSC development may provide therapeutic benefit. The pro-inflammatory molecules S100A8/A9 and high mobility group box protein 1 (HMGB1) and their receptor RAGE are strongly associated with the initiation and progression of most cancers. This article summarizes the literature demonstrating that these molecules are integrally involved in the early development, accumulation, and suppressive activity of MDSCs, and postulates that S100A8/A9 and HMGB1 serve as early biomarkers of disease and in conjunction with RAGE are potential targets for reducing MDSC levels and enhancing CBI and CAR-T immunotherapies.

The receptor for advanced glycation endproducts (RAGE) decreases survival of tumor-bearing mice by enhancing the generation of lung metastasis-associated myeloid-derived suppressor cells.

Metastatic cancer has a poor prognosis. Novel pharmacologic targets need to be identified. The receptor for advanced glycation endproducts (RAGE) is a pattern recognition receptor constitutively expressed in the lungs. Absence of overt disease in RAGE null mice suggests that RAGE is unnecessary or redundant in health. We report that RAGE null tumor-bearing mice have reduced lung metastasis and improved survival. Bone marrow chimera studies suggest that hematopoietic cell RAGE is an important contributor to these effects. Deletion of RAGE reduces both the quantity and suppressive activity of tumor-induced MDSC. Protein and mRNA studies suggest that RAGE contributes to the generation and function of MDSC including expression of the alarmins S100A8/A9 and activity of inducible nitric oxide synthase, arginase-1, and NF-κB. These findings demonstrate the important role of RAGE in determining the quantity and function of tumor-associated MDSC and suggest RAGE as a pharmacologic target for patients with metastatic disease.

Molecular cargo in myeloid-derived suppressor cells and their exosomes.

Collaborative research is reviewed in which mass spectrometry-based proteomics and next generation sequencing were used qualitatively and quantitatively to interrogate proteins and RNAs carried in intact myeloid-derived suppressor cells (MDSC) and exosomes shed in vitro by MDSC. In aggregate exosomes more than 4000 proteins were identified, including annexins and immunosuppressive mediators. Bioassays showed that exosomes induce MDSC chemotaxis dependent on S100A8 and S100A9 in their cargo. Surface selective chemistry identified glycoproteins on MDSC and exosome surfaces, including CD47 and thrombospondin 1, which both facilitate exosome-catalyzed chemotaxis. Large numbers of mRNAs and microRNAs were identified in aggregate exosomes, whose potential functions in receptor cells include angiogenesis, and proinflammatory and immunosuppressive activities. Inflammation was found to have asymmetric effects on MDSC and exosomal cargos. Collectively, our findings indicate that the exosomes shed by MDSC provide divergent and complementary functions that support the immunosuppression and tumor promotion activities of MDSC.

Therapies for tuberculosis and AIDS: myeloid-derived suppressor cells in focus.

The critical role of suppressive myeloid cells in immune regulation has come to the forefront in cancer research, with myeloid-derived suppressor cells (MDSCs) as a main oncology immunotherapeutic target. Recent improvement and standardization of criteria classifying tumor-induced MDSCs have led to unified descriptions and also promoted MDSC research in tuberculosis (TB) and AIDS. Despite convincing evidence on the induction of MDSCs by pathogen-derived molecules and inflammatory mediators in TB and AIDS, very little attention has been given to their therapeutic modulation or roles in vaccination in these diseases. Clinical manifestations in TB are consequences of complex host-pathogen interactions and are substantially affected by HIV infection. Here we summarize the current understanding and knowledge gaps regarding the role of MDSCs in HIV and Mycobacterium tuberculosis (co)infections. We discuss key scientific priorities to enable application of this knowledge to the development of novel strategies to improve vaccine efficacy and/or implementation of enhanced treatment approaches. Building on recent findings and potential for cross-fertilization between oncology and infection biology, we highlight current challenges and untapped opportunities for translating new advances in MDSC research into clinical applications for TB and AIDS.

Survival of the fittest: how myeloid-derived suppressor cells survive in the inhospitable tumor microenvironment.

Myeloid-derived suppressor cells (MDSC) are present in most cancer patients where they are significant contributors to the immune suppressive tumor microenvironment (TME). The TME is a hostile locale due to deficiencies in oxygen (hypoxia) and nutrients, and the presence of reactive oxygen species (ROS). The survival of tumor cells within the TME is partially governed by two mechanisms: (1) Activation of the transcription factor Nuclear Factor Erythroid-derived 2-like 2 (Nrf2) which turns on genes that attenuate oxidative stress; and (2) The presence of High Mobility Group Box Protein-1 (HMGB1), a damage-associated molecular pattern molecule (DAMP) that induces autophagy and protects against apoptosis. Because Nrf2 and HMGB1 promote tumor cell survival, we speculated that Nrf2 and HMGB1 may facilitate MDSC survival. We tested this hypothesis using Nrf2+/+ and Nrf2-/- BALB/c and C57BL/6 mice and pharmacological inhibitors of HMGB1. In vitro and in vivo studies demonstrated that Nrf2 increased the suppressive potency and quantity of tumor-infiltrating MDSC by up-regulating MDSC production of H2O2 and decreasing MDSC apoptosis. Decreased apoptosis was accompanied by a decrease in the production of MDSC, demonstrating that MDSC levels are homeostatically regulated. Pharmacological inhibition of autophagy increased MDSC apoptosis, indicating that autophagy increases MDSC half-life. Inhibition of HMGB1 also increased MDSC apoptosis and reduced MDSC autophagy. These results combined with our previous findings that HMGB1 drives the accumulation of MDSC demonstrate that HMGB1 maintains MDSC viability by inducing autophagy. Collectively, these findings identify Nrf2 and HMGB1 as important factors that enable MDSC to survive in the TME.

Top-Down Proteomic Characterization of Truncated Proteoforms.

A top-down proteomic strategy with semiautomated analysis of data sets has proven successful for the global identification of truncated proteins without the use of chemical derivatization, enzymatic manipulation, immunoprecipitation, or other enrichment. This approach provides the reliable identification of internal polypeptides formed from precursor gene products by proteolytic cleavage of both the N- and C-termini, as well as truncated proteoforms that retain one or the other termini. The strategy has been evaluated by application to the immunosuppressive extracellular vesicles released by myeloid-derived suppressor cells. More than 1000 truncated proteoforms have been identified, from which binding motifs are derived to allow characterization of the putative proteases responsible for truncation.

Differential Regulation of T-cell Immunity and Tolerance by Stromal Laminin Expressed in the Lymph Node.

BACKGROUND: Stromal laminins α4 and α5 are differentially regulated in transplant tolerance and immunity, respectively, resulting in altered T-cell trafficking. We hypothesized that laminins directly regulated T-cell activation and polarization. METHODS: Human and mouse CD4 T cells were activated in Th1, Th2, Th17, or regulatory T cell (Treg) environments with/without laminin α4 and/or α5. Laminin α5 receptors were blocked with anti-α6 integrin or anti-α-dystroglycan (αDG) monoclonal antibodies, and T-cell polarization was determined. T-cell receptor transgenic TEa CD4 cells that recognized donor alloantigen were transferred into C57BL/6 mice that received alloantigen or cardiac allografts. Laminin receptors were blocked, and TEa T-cell migration and differentiation were assessed. Laminin expression was measured in several models of immunity and tolerance. RESULTS: In diverse models, laminins α4 and α5 were differentially regulated. Immunity was associated with decreased laminin α4:α5 ratio, while tolerance was associated with an increased ratio. Laminin α4 inhibited CD4+ T-cell proliferation and Th1, Th2, and Th17 polarization but favored Treg induction. Laminin α5 favored T-cell activation and Th1, Th2, and Th17 polarization and inhibited Treg. Laminin α5 was recognized by T cell integrin α6 and is important for activation and inhibition of Treg. Laminin α5 was also recognized by T cell α-DG and required for Th17 differentiation. Anti-α6 integrin or anti-DG prolonged allograft survival. CONCLUSIONS: Laminins α4 and α5 are coinhibitory and costimulatory ligands for human and mouse CD4 T cells, respectively. Laminins and their receptors modulate immune responses by acting as one of the molecular switches for immunity or suppression.

Myeloid-Derived Suppressor Cells: Not Only in Tumor Immunity.

Since the realization that immature myeloid cells are powerful modulators of the immune response, many studies on "myeloid-derived suppressor cells" (MDSCs) have documented their ability to promote tumor progression in melanoma and other cancers. Whether MDSCs are induced solely pathologically in tumorigenesis, or whether they also represent physiological immune control mechanisms, is not well-understood, but is particularly important in the light of ongoing attempts to block their activities in order to enhance anti-tumor immunity. Here, we briefly review studies which explore (1) how best to identify MDSCs in the context of cancer and how this compares to other conditions in humans; (2) what the suppressive mechanisms of MDSCs are and how to target them pharmacologically; (3) whether levels of MDSCs with various phenotypes are informative for clinical outcome not only in cancer but also other diseases, and (4) whether MDSCs are only found under pathological conditions or whether they also represent a physiological regulatory mechanism for the feedback control of immunity. Studies unequivocally document that MDSCs strongly influence cancer outcomes, but are less informative regarding their relevance to infection, autoimmunity, transplantation and aging, especially in humans. So far, the results of clinical interventions to reverse their negative effects in cancer have been disappointing; thus, developing differential approaches to modulate MSDCs in cancer and other diseases without unduly comprising any normal physiological function requires further exploration.

Radiotherapy Both Promotes and Inhibits Myeloid-Derived Suppressor Cell Function: Novel Strategies for Preventing the Tumor-Protective Effects of Radiotherapy.

Cancer immunotherapies aimed at neutralizing the programmed death-1 (PD-1) immune suppressive pathway have yielded significant therapeutic efficacy in a subset of cancer patients. However, only a subset of patients responds to antibody therapy with either anti-PD-1 or anti-PD-L1 antibodies. These patients appear to have so-called "hot" tumors containing tumor-reactive T cells. Therefore, checkpoint blockade therapy may be effective in a larger percentage of cancer patients if combined with therapeutics that also activate tumor-reactive T cells. Radiotherapy (RT) is a prime candidate for combination therapy because it facilitates activation of both local antitumor immunity and antitumor immunity at non-radiated, distant sites (abscopal response). However, RT also promotes tumor cell expression of PD-L1 and facilitates the development of myeloid-derived suppressor cells (MDSC), a population of immune suppressive cells that also suppress through PD-L1. This article will review how RT induces MDSC, and then describe two novel therapeutics that are designed to simultaneously activate tumor-reactive T cells and neutralize PD-1-mediated immune suppression. One therapeutic, a CD3xPD-L1 bispecific T cell engager (BiTE), activates and targets cytotoxic T and NKT cells to kill PD-L1+ tumor cells, despite the presence of MDSC. The BiTE significantly extends the survival time of humanized NSG mice reconstituted with human PBMC and carrying established metastatic human melanoma tumors. The second therapeutic is a soluble form of the costimulatory molecule CD80 (sCD80). In addition to costimulating through CD28, sCD80 inhibits PD-1 suppression by binding to PD-L1 and sterically blocking PD-L1/PD-1 signaling. sCD80 increases tumor-infiltrating T cells and significantly extends survival time of mice carrying established, syngeneic tumors. sCD80 does not suppress T cell function via CTLA-4. These studies suggest that the CD3xPD-L1 BiTE and sCD80 may be efficacious therapeutics either as monotherapies or in combination with other therapies such as radiation therapy for the treatment of cancer.

Understanding the tumor immune microenvironment (TIME) for effective therapy.

The clinical successes in immunotherapy have been both astounding and at the same time unsatisfactory. Countless patients with varied tumor types have seen pronounced clinical response with immunotherapeutic intervention; however, many more patients have experienced minimal or no clinical benefit when provided the same treatment. As technology has advanced, so has the understanding of the complexity and diversity of the immune context of the tumor microenvironment and its influence on response to therapy. It has been possible to identify different subclasses of immune environment that have an influence on tumor initiation and response and therapy; by parsing the unique classes and subclasses of tumor immune microenvironment (TIME) that exist within a patient's tumor, the ability to predict and guide immunotherapeutic responsiveness will improve, and new therapeutic targets will be revealed.

Myeloid derived-suppressor cells: their role in cancer and obesity.

Myeloid-derived suppressor cells (MDSC) are present in most individuals with cancer where they inhibit adaptive and innate antitumor immunity and are an obstacle to cancer immunotherapies. Chronic inflammation is characteristic of adipose tissue and is a risk factor for the onset and progression of cancer in obese individuals. Because MDSC accumulate in response to inflammation, it has been hypothesized that one of the mechanisms by which obesity promotes malignancy is through the induction of MDSC. This article reviews the data supporting this hypothesis, the role of leptin and fatty acid metabolism in the induction of MDSC, and the surprising finding that although MDSC promote tumor progression, they are protective against some of the metabolic dysfunction associated with obesity.

Myeloid-Derived Suppressor Cells: Immune-Suppressive Cells That Impair Antitumor Immunity and Are Sculpted by Their Environment.

Myeloid-derived suppressor cells (MDSC) are a diverse population of immature myeloid cells that have potent immune-suppressive activity. Studies in both mice and humans have demonstrated that MDSC accumulate in most individuals with cancer, where they promote tumor progression, inhibit antitumor immunity, and are an obstacle to many cancer immunotherapies. As a result, there has been intense interest in understanding the mechanisms and in situ conditions that regulate and sustain MDSC, and the mechanisms MDSC use to promote tumor progression. This article reviews the characterization of MDSC and how they are distinguished from neutrophils, describes the suppressive mechanisms used by MDSC to mediate their effects, and explains the role of proinflammatory mediators and the tumor microenvironment in driving MDSC accumulation, suppressive potency, and survival.

Frontline Science: High fat diet and leptin promote tumor progression by inducing myeloid-derived suppressor cells.

Obesity is a risk factor for cancer incidence and cancer mortality. The association of obesity and cancer is attributed to multiple factors, but the tightest linkage is with the chronic, low-grade inflammation that accompanies obesity. Myeloid-derived suppressor cells (MDSC) are known facilitators of cancer progression that act by suppressing the activation and function of tumor-reactive T cells. Because MDSC quantity and function are driven by chronic inflammation, we hypothesized that MDSC may accumulate in obese individuals and facilitate tumor growth by suppressing antitumor immunity. To test this hypothesis, tumor-bearing mice on a high fat or low fat diet (HFD or LFD) were assessed for tumor progression and the metabolic dysfunction associated with obesity. HFD enhanced the accumulation of MDSC, and the resulting MDSC had both beneficial and detrimental effects. HFD-induced MDSC protected mice against diet-induced metabolic dysfunction and reduced HFD-associated inflammation, but also increased the accumulation of fat, enhanced tumor progression, and spontaneous metastasis and reduced survival time. HFD-induced MDSC facilitated tumor growth by limiting the activation of tumor-reactive CD8+ T cells. Leptin, an adipokine that regulates appetite satiety and is overexpressed in obesity, undergoes crosstalk with MDSC in which leptin drives the accumulation of MDSC while MDSC down-regulate the production of leptin. Collectively, these studies demonstrate that although MDSC protect against some metabolic dysfunction associated with HFD they enhance tumor growth in HFD mice and that leptin is a key regulator linking HFD, chronic inflammation, immune suppression, and tumor progression.

Soluble CD80 Protein Delays Tumor Growth and Promotes Tumor-Infiltrating Lymphocytes.

Tumor cells use various immune-suppressive strategies to overcome antitumor immunity. One such method is tumor expression of programmed death ligand-1 (PD-L1), which triggers apoptotic death or anergy upon binding programmed death-1 (PD-1) on T cells. Our previous in vitro cellular studies with human and mouse PD-L1+ tumor cells demonstrated that a soluble form of the costimulatory molecule CD80 prevented PD-L1-mediated immune suppression and restored T-cell activation by binding PD-L1 and blocking interaction with PD-1. We now report that in vivo treatment of established syngeneic PD-L1+ CT26 colon carcinoma and B16F10 melanoma tumors with CD80-Fc delays tumor growth and promotes tumor-infiltrating T cells. Studies with PD-1-/- and CD28-/- mice demonstrate that soluble CD80 acts in vivo by simultaneously neutralizing PD-1 suppression and activating through CD28. We also report that soluble CD80 mediates its effects by activating transcription factors EGR1-4, NF-κB, and MAPK, downstream signaling components of the CD28 and T-cell receptor pathways. Soluble CD80 binds to CTLA-4 on activated human peripheral blood mononuclear cells. However, increasing quantities of CTLA-4 antagonist antibodies do not increase T-cell activation. These results indicate that soluble CD80 does not suppress T-cell function through CTLA-4 and suggest that CTLA-4 acts as a decoy receptor for CD80, rather than functioning as a suppressive signaling receptor. Collectively, these studies demonstrate that soluble CD80 has therapeutic efficacy in vivo in mouse tumor systems and that its effects are due to its ability to inhibit PD-1-mediated suppression while concurrently activating T cells through CD28. Cancer Immunol Res; 6(1); 59-68. ©2017 AACR.

Ubiquitin Conjugation Probed by Inflammation in Myeloid-Derived Suppressor Cell Extracellular Vesicles.

Ubiquitinated proteins carried by the extracellular vesicles (EV) released by myeloid-derived suppressor cells (MDSC) have been investigated using proteomic strategies to examine the effect of tumor-associated inflammation. EV were collected from MDSC directly following isolation from tumor-bearing mice with low and high inflammation. Among the 1092 proteins (high inflammation) and 925 proteins (low inflammation) identified, more than 50% were observed as ubiquitinated proteoforms. More than three ubiquitin-attachment sites were characterized per ubiquitinated protein, on average. Multiple ubiquitination sites were identified in the pro-inflammatory proteins S100 A8 and S100 A9, characteristic of MDSC and in histones and transcription regulators among other proteins. Spectral counting and pathway analysis suggest that ubiquitination occurs independently of inflammation. Some ubiquitinated proteins were shown to cause the migration of MDSC, which has been previously connected with immune suppression and tumor progression. Finally, MDSC EV are found collectively to carry all the enzymes required to catalyze ubiquitination, and the hypothesis is presented that a portion of the ubiquitinated proteins are produced in situ.

Differential Content of Proteins, mRNAs, and miRNAs Suggests that MDSC and Their Exosomes May Mediate Distinct Immune Suppressive Functions.

Myeloid-derived suppressor cells (MDSC) are immature myeloid cells that accumulate in the circulation and the tumor microenvironment of most cancer patients. There, MDSC suppress both adaptive and innate immunity, hindering immunotherapies. The inflammatory milieu often present in cancers facilitates MDSC suppressive activity, causing aggressive tumor progression and metastasis. MDSC from tumor-bearing mice release exosomes, which carry biologically active proteins and mediate some of the immunosuppressive functions characteristic of MDSC. Studies on other cell types have shown that exosomes may also carry RNAs which can be transferred to local and distant cells, yet the mRNA and microRNA cargo of MDSC-derived exosomes has not been studied to date. Here, the cargo of MDSC and their exosomes was interrogated with the goal of identifying and characterizing molecules that may facilitate MDSC suppressive potency. Because inflammation is an established driving force for MDSC suppressive activity, we used the well-established 4T1 mouse mammary carcinoma system, which includes "conventional" as well as "inflammatory" MDSC. We provide evidence that MDSC-derived exosomes carry proteins, mRNAs, and microRNAs with different quantitative profiles than those of their parental cells. Several of these molecules have known or predicted functions consistent with MDSC suppressive activity, suggesting a potential mechanistic redundancy.

CD3xPDL1 bi-specific T cell engager (BiTE) simultaneously activates T cells and NKT cells, kills PDL1+ tumor cells, and extends the survival of tumor-bearing humanized mice.

Bi-specific T cell engagers (BiTEs) activate T cells through CD3 and target activated T cells to tumor-expressed antigens. BiTEs have shown therapeutic efficacy in patients with liquid tumors; however, they do not benefit all patients. Anti-tumor immunity is limited by Programmed Death 1 (PD1) pathway-mediated immune suppression, and patients who do not benefit from existing BiTES may be non-responders because their T cells are anergized via the PD1 pathway. We have designed a BiTE that activates and targets both T cells and NKT cells to PDL1+ cells. In vitro studies demonstrate that the CD3xPDL1 BiTE simultaneously binds to both CD3 and PDL1, and activates healthy donor CD4+ and CD8+ T cells and NKT cells that are specifically cytotoxic for PDL1+ tumor cells. Cancer patients' PBMC are also activated and cytotoxic, despite the presence of myeloid-derived suppressor cells. The CD3xPDL1 BiTE significantly extends the survival time and maintains activated immune cell levels in humanized NSG mice reconstituted with human PBMC and carrying established human melanoma tumors. These studies suggest that the CD3xPDL1 BiTE may be efficacious for patients with PDL1+ solid tumors, in combination with other immunotherapies that do not specifically neutralize PD1 pathway-mediated immune suppression.