Myeloid-derived Suppressor Cells In Blood Research Articles

Increase in both CD14-positive and CD15-positive myeloid-derived suppressor cell subpopulations in the blood of patients with glioma but predominance of CD15-positive myeloid-derived suppressor cells in glioma tissue.

Myeloid-derived suppressor cells (MDSCs), defined as CD33-positive major histocompatibility complex class II-negative cells, are increased in a variety of human tumors and are associated with immunosuppression. Myeloid-derived suppressor cells can be further subdivided into CD14-positive monocytic MDSC and CD15-positive granulocytic MDSC (polymorphonuclear MDSC) subpopulations. Here we analyzed MDSC subsets in the blood and tumor tissue of patients with glioma, including the most malignant variant, glioblastoma multiforme (GBM). CD33-positive major histocompatibility complex class II-negative MDSCs in blood from 21 patients with glioma and 12 healthy individuals were phenotyped and quantified by flow cytometry. Myeloid populations of the monocytic MDSC and polymorphonuclear MDSC phenotypes were both significantly increased in the blood of patients with GBM versus healthy controls. The myeloid activation markers CD80 and PD-L1 could not be detected on either of these MDSC subsets; CD124, CD86, and CD40 were detected at similar levels on MDSCs in patients with glioma and healthy donors. By contrast, in tumor cell suspensions, the MDSC population consisted almost exclusively of CD15-positive cells. Immunohistochemistry confirmed infiltration of CD15-positive major histocompatibility complex class II-negative cells in glioma tissue samples. These data support a role for cells with an MDSC phenotype in the blood and tumor microenvironment of patients with GBM.

Sunitinib depletes myeloid-derived suppressor cells and synergizes with a cancer vaccine to enhance antigen-specific immune responses and tumor eradication

The high efficacy of therapeutic cancer vaccines in preclinical studies has yet to be fully achieved in clinical trials. Tumor immune suppression is a critical factor that hampers the desired antitumor effect. Here, we analyzed the combined effect of a cancer vaccine and the receptor tyrosine kinase inhibitor sunitinib. Sunitinib was administered intraperitoneally, alone or in combination with intramuscular immunization using a viral vector based cancer vaccine composed of Semliki Forest virus replicon particles and encoding the oncoproteins E6 and E7 (SFVeE6,7) of human papilloma virus (HPV). We first demonstrated that treatment of tumor-bearing mice with sunitinib alone dose-dependently depleted myeloid-derived suppressor cells (MDSCs) in the tumor, spleen and in circulation. Concomitantly, the number of CD8+ T cells increased 2–fold and, on the basis of CD69 expression, their activation status was greatly enhanced. The intrinsic immunosuppressive activity of residual MDSCs after sunitinib treatment was not changed in a dose-dependent fashion. We next combined sunitinib treatment with SFVeE6,7 immunization. This combined treatment resulted in a 1.5- and 3-fold increase of E7-specific cytotoxic T lymphocytes (CTLs) present within the circulation and tumor, respectively, as compared to immunization only. The ratio of E7-specific CTLs to MDSCs in blood thereby increased 10- to 20-fold and in tumors up to 12.5-fold. As a result, the combined treatment strongly enhanced the antitumor effect of the cancer vaccine. This study demonstrates that sunitinib creates a favorable microenvironment depleted of MDSCs and acts synergistically with a cancer vaccine resulting in enhanced levels of active tumor-antigen specific CTLs, thus changing the balance in favor of antitumor immunity.

Tadalafil reduces myeloid-derived suppressor cells and regulatory T cells and promotes tumor immunity in patients with head and neck squamous cell carcinoma.

Myeloid-derived suppressor cells (MDSC) and regulatory T cells (Treg) play a key role in the progression of head and neck squamous cell carcinoma (HNSCC). On the basis of our preclinical data demonstrating that phosphodiesterase-5 (PDE5) inhibition can modulate these cell populations, we evaluated whether the PDE5 inhibitor tadalafil can revert tumor-induced immunosuppression and promote tumor immunity in patients with HNSCC. First, we functionally and phenotypically characterized MDSCs in HNSCCs and determined, retrospectively, whether their presence at the tumor site correlates with recurrence. Then, we performed a prospective single-center, double-blinded, randomized, three-arm study in which patients with HNSCC undergoing definitive surgical resection of oral and oropharyngeal tumors were treated with tadalafil 10 mg/day, 20 mg/day, or placebo for at least 20 days preoperatively. Blood and tumor MDSC and Treg presence and CD8(+) T-cell reactivity to tumor antigens were evaluated before and after treatment. MDSCs were characterized in HNSCC and their intratumoral presence significantly correlates with recurrence. Tadalafil treatment was well tolerated and significantly reduced both MDSCs and Treg concentrations in the blood and in the tumor (P < 0.05). In addition, the concentration of blood CD8(+) T cells reactive to autologous tumor antigens significantly increased after treatment (P < 0.05). Tadalafil immunomodulatory activity was maximized at an intermediate dose but not at higher doses. Mechanistic analysis suggests a possible off-target effect on PDE11 at high dosages that, by increasing intracellular cAMP, may negatively affect antitumor immunity. Tadalafil seems to beneficially modulate the tumor micro- and macro-environment in patients with HNSCC by lowering MDSCs and Tregs and increasing tumor-specific CD8(+) T cells in a dose-dependent fashion.

Reduced Myeloid-derived Suppressor Cells in the Blood and Endometrium is Associated with Early Miscarriage

The contribution of myeloid-derived suppressor cells (MDSC) in patients suffering from early or recurrent miscarriage is unknown. MDSC are implicated in modulation of T-cell response in healthy pregnancies; however, the role of MDSC in patients suffering from miscarriage has not been studied. We hypothesized that MDSC play major role in inducing maternal-fetal tolerance and this tolerance is compromised in patients suffering from miscarriage. MDSC level was assessed by flow cytometry and immunostaining in blood and endometrial decidua, respectively. Activation of T cells was determined by MTT proliferation and IL-2 ELISA assays. The miscarriage patients harbor reduced level of functionally suppressive MDSC in blood and endometrium as compared to healthy control women with successful pregnancies. These results suggest MDSC regulate maternal tolerance in healthy pregnancies and that drug inducing MDSC could have therapeutic implication in the miscarriage patients.

Myeloid-derived suppressor cells have a proinflammatory role in the pathogenesis of autoimmune arthritis

ObjectivesAlthough myeloid-derived suppressor cells (MDSCs) have been linked to T cell tolerance, their role in autoimmune rheumatoid arthritis (RA) remains elusive. Here we investigate the potential association of MDSCs with...

Myeloid derived suppressor and dendritic cell subsets are related to clinical outcome in prostate cancer patients treated with prostate GVAX and ipilimumab.

BackgroundCancer-related disturbances in myeloid lineage development, marked by high levels of myeloid-derived suppressor cells (MDSC) and impaired dendritic cell (DC) development, are associated with poor clinical outcome due to immune escape and therapy resistance. Redressing this balance may therefore be of clinical benefit. Here we investigated the effects of combined Prostate GVAX/ipilimumab immunotherapy on myeloid subsets in peripheral blood of castration-resistant prostate cancer (CRPC) patients as well as the putative predictive value of baseline and on-treatment myeloid parameters on clinical outcome.MethodsPatients with CRPC (n = 28) received thirteen intradermal administrations of Prostate GVAX, consisting of two allogeneic GM-CSF-transduced and irradiated prostate cancer cell lines (LN-CaP and PC3) and six infusions of escalating doses of anti-CTLA4/ipilimumab. Frequencies and activation status of peripheral blood DC (PBDC) and MDSC were determined before, during and after treatment by flowcytometric analysis and related to clinical benefit.ResultsSignificant treatment-induced activation of conventional and plasmacytoid DC subsets (cDC and pDC) was observed, which in the case of BDCA1/CD1c+ cDC1 and MDC8+/6-sulfoLacNAc+ inflammatory cDC3 was associated with significantly prolonged overall survival (OS), but also with the development of autoimmune-related adverse events. High pre-treatment levels of CD14+HLA-DR−monocytic MDSC (mMDSC) were associated with reduced OS. Unsupervised clustering of these myeloid biomarkers revealed particular survival advantage in a group of patients with high treatment-induced PBDC activation and low pretreatment frequencies of suppressive mMDSC in conjunction with our previously identified lymphoid biomarker of high pretreatment CD4+CTLA4+ T cell frequencies.ConclusionsOur data demonstrate that DC and MDSC subsets are affected by prostate GVAX/ipilimumab therapy and that myeloid profiling may contribute to the identification of patients with possible clinical benefit of Prostate GVAX/ipilimumab treatment.Electronic supplementary materialThe online version of this article (doi:10.1186/s40425-014-0031-3) contains supplementary material, which is available to authorized users.

The Role of Myeloid-Derived Suppressor Cells in Immune Ontogeny

Myeloid-derived suppressor cells (MDSC) are a heterogeneous population of granulocytic or monocytic cells that suppress innate as well as adaptive immune responses. In healthy adults, immature myeloid cells differentiate into macrophages, dendritic cells, and granulocytes in the bone marrow and MDSC are rarely detected in peripheral blood. However, in certain pathologies, in particular malignancies and chronic infection, differentiation of these cells is altered resulting in accumulation of circulating suppressive myeloid cells. MDSC express suppressive factors such as arginase-1, reactive oxygen species, and inducible nitric oxide synthase, which have the ability to inhibit T cell proliferation and cytoxicity, induce the expansion of regulatory T cells, and block natural killer cell activation. It is increasingly recognized that MDSC alter the immune response to several cancers, and perhaps chronic viral infections, in clinically important ways. In this review, we outline the potential contribution of MDSC to the generation of feto-maternal tolerance and to the ineffective immune responses to many infections and vaccines observed in early post-natal life. Granulocytic MDSC are present in large numbers in pregnant women and in cord blood, and wane rapidly during infancy. Furthermore, cord blood MDSC suppress in vitro T cell and NK responses, suggesting that they may play a significant role in human immune ontogeny. However, there are currently no data that demonstrate in vivo effects of MDSC on feto-maternal tolerance or immune ontogeny. Studies are ongoing to evaluate the functional importance of MDSC, including their effects on control of infection and response to vaccination in infancy. Importantly, several pharmacologic interventions have the potential to reverse MDSC function. Understanding the role of MDSC in infant ontogeny and their mechanisms of action could lead to interventions that reduce mortality due to early-life infections.

Generation of a new therapeutic peptide that depletes myeloid-derived suppressor cells in tumor-bearing mice

Cancer immune evasion is an emerging hallmark of disease progression. Functional studies to understand the role of myeloid-derived suppressor cells (MDSC) in the tumor microenvironment however, are limited by the lack of available specific cell surface markers. We adapted a competitive peptide phage display platform to identify candidate peptides binding MDSC specifically and generated peptide-Fc fusion proteins (peptibody). In multiple tumor models peptibody injection iv completely depleted blood, splenic, and intratumoral MDSC in tumor-bearing mice, without affecting proinflammatory immune cell types, such as dendritic cells. While control Gr-1 antibody depleted primarily granulocytic MDSC, peptibodies depleted both granulocytic and monocytic subsets. Remarkably, peptibody treatment was associated with inhibition of tumor growth in vivo, which was superior to Gr-1. Immunoprecipitation of MDSC membrane proteins identified S100 family proteins as candidate targets. Our strategy may be useful to identify novel diagnostic and therapeutic surface targets on rare cell subtypes, including human MDSC.

Myeloid expression of angiotensin-converting enzyme facilitates myeloid maturation and inhibits the development of myeloid-derived suppressor cells

Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of immature myeloid cells which accumulate in cancer, infection and chronic inflammation. These cells suppress T-cell function and the immune response. Angiotensin-converting enzyme (ACE) is a peptidase that is now known to regulate aspects of myelopoiesis. Here, we show that ACE expression correlates with myeloid maturation in vitro. Forced ACE overexpression in monocytic cells reduces the generation of MDSCs. In vivo, mice with a genetic change resulting in myeloid cell ACE overexpression have reduced numbers of blood and splenic MDSCs in a tumor model and in a model of chronic inflammation induced by complete Freund's adjuvant. In contrast, ACE-null mice produce large numbers of MDSCs during chronic inflammation. Macrophages from mice with myeloid ACE overexpressing are more pro-inflammatory and have more tumor-killing activity than cells from wild-type mice. Thus, manipulating myeloid ACE activity can interfere with MDSC development and the maturation of myeloid cells.

Adults with indolent systemic mastocytosis have elevated peripheral blood myeloid-derived suppressor cells compared to chronic urticaria and healthy controls. (HUM7P.320)

Abstract MDSCs are a heterogeneous population of immature myeloid cells shown to be immunomodulatory in many diseases. In humans, they are identified by surface expression of CD33 and CD11b and lack of HLA DR. MDSCs are further divided into monocytic (M-MDSCs, CD14+) and granulocytic (G-MDSCs, CD15+) subsets. Our previous data showed that the prevalence and suppressive activity of murine MDSCs was augmented by the presence of mast cells (MCs). Indolent systemic mastocytosis (ISM) is a human disease caused by the clonal expansion of MCs. We propose that increased MCs and MC mediators frequently associated with mastocytosis produce an increase in peripheral blood MDSCs. Peripheral blood mononuclear cells (PBMCs) were isolated from blood obtained from adults with ISM and from chronic urticaria (CU, drug control), and healthy (H) age-matched controls. PBMCs, labeled with antibodies against HLA-DR, CD33, CD11b, CD15, and CD14, were analyzed by flow cytometry. MDSC concentrations in blood (x 106 cells/mL, mean ± standard error) were calculated. MDSC concentration was elevated (p&amp;lt;0.0001) in ISM (15±7) versus CU (1.4±0.4) and H (2.8±0.4). The G-MDSC subset (9.4±4, 0.6±0.1, 1.5±0.3 for ISM, CU, and H respectively, p&amp;lt;0.001) largely accounted for this elevation. A similar, non-significant trend (p=0.24) is seen in the M-MDSCs (1.6±0.5, 0.4±0.1, 0.7±0.3). Overall, these results demonstrate that MDSCs, particularly G-MDSCs, are elevated in the blood of patients with ISM, warranting further study.

Myeloid-derived suppressor cells are increased in frequency but not maximally suppressive in peripheral blood of Type 1 Diabetes Mellitus patients.

Type 1 Diabetes Mellitus (T1D) results from the destruction of insulin-producing beta cells in the pancreas by autoreactive T cells. Myeloid derived suppressor cells (MDSCs) are a recently identified immune cell subset that down-regulate T cells. Whether defects in MDSC numbers or function may contribute to T1D pathogenesis is not known. We report here that MDSCs are unexpectedly enriched in peripheral blood of both mice and patients with autoimmune diabetes. Peripheral blood MDSCs from T1D patients suppressed T cell proliferation in a contact-dependent manner; however, suppressive function could be enhanced with in vitro cytokine induction. These findings suggest that native T1D MDSCs are not maximally suppressive and that strategies to promote MDSC suppressive function may be effective in preventing or treating T1D.

Specific Inhibition of the VEGFR-3 Tyrosine Kinase by SAR131675 Reduces Peripheral and Tumor Associated Immunosuppressive Myeloid Cells

Myeloid derived suppressor cells (MDSCs) and tumor-associated macrophages (TAMs) represent prominent components in cancer progression. We previously showed that inhibition of the VEGFR-3 pathway by SAR131675 leads to reduction of TAM infiltration and tumor growth. Here, we found that treatment with SAR131675 prevents the accumulation of immunosuppressive blood and splenic MDSCs which express VEGFR-3, in 4T1 tumor bearing mice. Moreover we showed that soluble factors secreted by tumor cells promote MDSCs proliferation and differentiation into M2 polarized F4/80+ macrophages. In addition, cell sorting and transcriptomic analysis of tumor infiltrating myeloid cells revealed the presence of a heterogeneous population that could be divided into 3 subpopulations: (i) immature cells with a MDSC phenotype (GR1+/CD11b+/F4/80−); (ii) “immuno-incompetent” macrophages (F4/80high/CD86neg/MHCIILow) strongly expressing M2 markers such as Legumain, CD206 and Mgl1/2 and (iii) “immuno-competent”-M1 like macrophages (F4/80Low/CD86+/MHCIIHigh). SAR131675 treatment reduced MDSCs in lymphoid organs as well as F4/80High populations in tumors. Interestingly, in the tumor SAR131675 was able to increase the immunocompetent M1 like population (F4/80low). Altogether these results demonstrate that the specific VEGFR-3 inhibitor SAR131675 exerts its anti tumoral activity by acting on different players that orchestrate immunosuppression and cancer progression in a tumoral context: MDSCs in peripheral lymphoid organs and TAMs infiltrating the tumor.

Silibinin inhibits accumulation of myeloid-derived suppressor cells and tumor growth of murine breast cancer.

Myeloid-derived suppressor cells (MDSC)s increase in blood and accumulate in the tumor microenvironment of tumor-bearing animals, contributing to immune suppression in cancer. Silibinin, a natural flavonoid from the seeds of milk thistle, has been developed as an anti-inflammatory agent and supportive care agent to reduce the toxicity of cancer chemotherapy. The goals of this study were to evaluate the effect of silibinin on MDSCs in tumor-bearing mice and antitumor activity of silibinin in a mouse model of breast cancer. 4T1 luciferase-transfected mammary carcinoma cells were injected into in the mammary fat pad female BALB/c mice, and female CB17-Prkdc Scid/J mice. Silibinin treatment started on day 4 or day 14 after tumor inoculation continued every other day. Tumor growth was monitored by bioluminescent imaging (BLI) measuring total photon flux. Flow cytometry measured total leukocytes, CD11b+ Gr-1+ MDSC, and T cells in the blood and tumors of tumor-bearing mice. The effects of silibinin on 4T1 cell viability in vitro were measured by BLI. Treatment with silibinin increased overall survival in mice harboring tumors derived from the 4T1-luciferase breast cancer cell line, and reduced tumor volumes and numbers of CD11b+Gr-1+ MDSCs in the blood and tumor, and increased the content of T cells in the tumor microenvironment. Silibinin failed to inhibit tumor growth in immunocompromised severe combined immunodeficiency mice, supporting the hypothesis that anticancer effect of silibinin is immune-mediated. The antitumor activity of silibinin requires an intact host immune system and is associated with decreased accumulation of blood and tumor-associated MDSCs.

Targeted TLR9-activation/STAT3-blocking abrogates immunosuppressive functions of myeloid-derived suppressor cells from late-stage prostate cancer patients

Recent clinical advances underscored that elimination of tumor-induced immunosuppression is prerequisite for successful therapy of advanced human cancers. The myeloid-derived suppressor cells (MDSCs) are one of the major populations limiting T cell-mediated antitumor immune responses in late-stage solid tumors, such as castration-resistant prostate cancers (CRPCs). Targeting MDSCs proved challenging due to their heterogeneity and phenotypic similarities with other myeloid cell lineages. Here, we identify a population of granulocytic MDSCs, which accumulate in patients' blood during progression of prostate cancer from localized to metastatic disease. The CRPC-associated MDSCs potently inhibit proliferation, IFN-γ and Granzyme-B production in autologous CD8+ T cells. Importantly, the MDSCs in blood and in lymph nodes of CRPC patients show high levels of activated STAT3 transcription factor, which is a crucial immune checkpoint regulator. We previously generated an original method to silence genes specifically in TLR9+ myeloid cells using CpG-siRNA conjugates [1,2]. Now, we demonstrate that the CpG-STAT3siRNA is also effectively internalized by human MDSCs and induces STAT3 silencing without the need for transfection reagents. Our further studies demonstrated that two-pronged TLR9-activation/STAT3-blocking by CpG-STAT3siRNA inhibits immunosuppressive effects of CRPC-MDSCs on CD8+ T cells, while both TLR9 triggering or STAT3 inhibition alone have only minimal effects. The reversal of MDSC immunosuppression by CpG-STAT3 siRNA results from reduced expression and enzymatic activity of Arginase I, a downstream STAT3 target gene. Overall, our study demonstrates the feasibility of overcoming immunosuppression in CRPC patients using CpG-STAT3siRNA strategy to alleviate effects of MDSCs. We anticipate that our strategy to reverse MDSC-mediated immunosuppression without depleting myeloid cell populations will generate safer and more effective immunotherapy for late-stage, metastatic prostate cancers and potentially other solid tumors. This project described was supported by the National Cancer Institute of the National Institutes of Health under award number R01CA155367 and by the Department of Defense Prostate Cancer Research Program under award number W81XWH-12-1-0132 to M.K.

Granulocytic myeloid-derived suppressor cells expand in cord blood and human pregnancy and modulate T cell responses

Background Preterm delivery is a leading cause of perinatal morbidity. Reasons are diverse, but immunologic fetal rejection has repeatedly been postulated to be involved. Myeloid derived suppressor cells (MDSC) are myeloid progenitor cells, characterized by their T cell suppressive capacity. They have predominantly been described under pathological conditions like cancer or infectious diseases, yet their role for materno-fetal tolerance remains elusive. We aimed to quantify and characterize MDSC in cord blood (CB) and in pregnancy.

Association of Myeloid-derived Suppressor Cells and Efficacy of Cytokine-induced Killer Cell Immunotherapy in Metastatic Renal Cell Carcinoma Patients

Metastatic renal cell carcinoma (MRCC) is one of the malignancies that are sensitive to immunotherapy. However, the underlying immune inhibitory factors such as myeloid-derived suppressor cells (MDSCs) might restrain the efficacy of immunotherapy. The present study investigates the clinical efficacy of cytokine-induced killer (CIK) cell therapy in patients with MRCC and explores whether the levels of peripheral MDSCs are associated with the prognosis of patients receiving this therapy. Twenty-nine patients with measurable MRCC were treated with an adoptive transfer of autologous CIK cells, followed by 5 consecutive days of interleukin-2 administration. The tumor response and 1-year survival were observed. The proportion of MDSCs in the peripheral blood was detected, and the correlation of MDSCs with prognosis was analyzed. Of 29 evaluable patients, no complete responses were seen; 4 patients exhibited a partial response (13.8%), 18 patients displayed stable disease (62.1%), and 7 patients showed progressive disease (24.1%). Twenty patients (69.0%) were alive 14.8-41.4 months at the time of the last follow-up (median follow-up=20.2 mo). The 1-year survival was 82.8% (24/29). Peripheral blood MDSCs were elevated in almost all MRCC patients and decreased after CIK-cell infusion. Subgroup analysis indicated that patients with a relatively low proportion of MDSCs exhibited prolonged survival. In conclusion, our data suggest that transfusion of autologous CIK cells can induce regression of MRCC, and MDSCs can serve as a potential marker for the prognosis of patients receiving a CIK-based therapy.

Amplification of functional myeloid-derived suppressor cells during stem cell mobilization induced by granulocyte colony-stimulation-factor

The effects of granulocyte colony-stimulation-factor (G-CSF) on stem cell mobilization and its impact on the amplification of myeloid-derived suppressor cells (MDSCs) of donor mice were examined. A mouse model of stem cell mobilization was established by consecutive subcutaneous injection of 100 μg/kg G-CSF for 5 days. The blood from the donor mice was routinely examined during mobilization. Stem cells and MDSCs were analyzed by flow cytometry. The immunosuppressive molecules derived from MDSCs in serum and spleen, including hydrogen dioxide (H2O2) and nitric oxide (NO), and the activity of nitric oxide synthase (NOS) were determined during the mobilization. Apoptosis of T lymphocytes was assessed by using Annexin-V/PI. During stem cell mobilization, the number of lymphocytes and white blood cells in the peripheral blood was increased, and peaked on the 4th day. The number of stem cells in G-CSF-treated mice was significantly greater than that in controls (P<0.01). The expansions of MSDCs were also observed after G-CSF mobilization, with a more notable rate of growth in the peripheral blood than in the spleen. The activity of NOS and the production of NO were increased in the donor mice, and the serum H2O2 levels were approximately 4-fold greater than the controls. Consequently, apoptosis of T lymphocytes was increased and showed a positive correlation with the elevated percentage of MDSCs. It was concluded that G-CSF could provide sufficient peripheral blood stem cells for transplantation. Exogenous administration of G-CSF caused the accumulation of MDSCs in the peripheral blood and the spleen, which could lead to apoptosis of T lymphocytes and may offer a new strategy for the prevention and treatment of graft versus host disease.

Significance of myeloid-derived suppressor cells in squamous cell carcinoma of the head and neck.

6070 Background: Patients with advanced stage squamous cell carcinoma of the head and neck (SCCHN) have less than 50% 5-year survival rate Human papillomavirus (HPV)-associated SCCHN in oropharyngeal sites have shown better prognosis. Little is known about the role of myeloid-derived suppressor cells (MDSCs) in immune suppression or tumor progression in the setting of SCCHN. Our objective is to evaluate the clinical significance of MDSCs in subjects with SCCHN, HPV-positivity, and advanced cancer staging. Methods: Thirty-three subjects with SCCHN and 10 healthy donors were enrolled in this prospective cohort study. Fresh blood was collected at the time of surgical resection of SCCHN in a tertiary academic center between August 2011 and January 2013. Peripheral blood mononuclear cells (PBMCs) were obtained using Ficoll Hypaque. MDSCs were immunophenotyped as CD14-CD33+CD11b+by flow cytometry. HPV status was determined by in situ hybridization Frequencies of MDSCs in blood of different cohorts were evaluated. Results: Thirty-three subjects (ages 34-83 years, 25 males) with SCCHN were enrolled. Increased numbers of CD14-CD33+CD11b+ cells of total leukocytes were found in HPV-associated SCCHN (median 26.6%, n=11) compared to HPV-negative SCCHN (16.3%, n=19). Interestingly, 3 subjects who previously had HPV-positive SCCHN but with no evidence of disease had 6.24% (n=3) CD14-CD33+CD11b+cells of leukocytes which was higher than healthy donors (3.55%, n=10). Subjects with advanced cancer stages (III-IV) had higher levels of MDSCs (26%, n=19) compared to those with a lower grade (I-II, 15.5%, n=11) regardless of HPV status. Three subjects were lost to follow up. Of the remaining subjects, the overall median follow time was 3 months and subjects who were found to have recurrence, regional or local metastasis had higher frequencies of MDSCs in the blood (26.35%, n=4) compared to those with no evidence of disease (18.5%, n=26) at the time of surgery. Conclusions: This study suggests there is an accumulation of MDSCs in peripheral blood of patients with SCCHN, particularly in HPV-associated SCCHN. Further, increased levels of MDSCs in the peripheral blood are related to more advance cancer stage and poor clinical outcomes.

Myeloid-derived suppressor cells have a central role in attenuated Listeria monocytogenes-based immunotherapy against metastatic breast cancer in young and old mice

Background:Myeloid-derived suppressor cells (MDSCs) are present in large numbers in blood of mice and humans with cancer, and they strongly inhibit T-cell and natural killer (NK) cell responses, at young and old age. We found that a highly attenuated bacterium Listeria monocytogenes (Listeriaat)-infected MDSC and altered the immune-suppressing function of MDSC.Methods:Young (3 months) and old (18 months) BALB/cByJ mice with metastatic breast cancer (4T1 model) were immunised with Listeriaat semi-therapeutically (once before and twice after tumour development), and analysed for growth of metastases and primary tumour, in relation to MDSC-, CD8 T-cell and NK cell responses.Results:We found that Listeriaat-infected MDSC, which delivered Listeriaat predominantly to the microenvironment of metastases and primary tumours, where they spread from MDSC into tumour cells (infected tumour cells will ultimately become a target for Listeria-activated immune cells). Immunotherapy with Listeriaat significantly reduced the population of MDSC in blood and primary tumours, and converted a remaining subpopulation of MDSC into an immune-stimulating phenotype producing IL-12, in correlation with significantly improved T-cell and NK cell responses to Listeriaat at both ages. This was accompanied with a dramatic reduction in the number of metastases and tumour growth at young and old age.Conclusions:Although preclinical studies show that immunotherapy is less effective at old than at young age, our study demonstrates that Listeriaat-based immunotherapy can be equally effective against metastatic breast cancer at both young and old age by targeting MDSC.

Abstract 4986: Myeloid-derived suppressor cells as a biomarker of tumor growth and radiosensitivity: Role of hypoxia-inducible arginase-1.

Abstract Introduction: Recent evidence suggests that tumor growth and metastases are sustained by myeloid-derived suppressor cells (MDSC), which over-express arginase-1 (ARG) and cause T-cell suppression. We hypothesize that MDSC impair tumor cell radiosensitivity through ARG-mediated depletion of L-arginine, an essential substrate for the biosynthesis of the endogenous radiosensitizer nitric oxide (NO). Aim: We explored whether MDSC and ARG can be used as biomarkers for tumor growth in experimental mouse models and in the clinical setting of colorectal cancer (CRC). We further examined whether low L-arginine levels are associated with an impaired generation of NO by inducible NO synthase (iNOS) and a reduced radiosensitivity of hypoxic tumor cells. Materials &amp; methods: MDSC (CD11b+Gr-1+) and ARG levels in mouse colon CT26 and mammary 4T1 models were analyzed by flow cytometry (FCM) and RT-PCR. Mice were inoculated I.M. with either CT26 or 4T1 carcinoma cells. After 17 days, MDSC were quantified in the spleen by FCM. In addition, ARG levels were analyzed by RT-PCR and FCM. Similar analyses were performed on human blood MDSC (CD14+HLA-DRlow) derived from CRC patients. To model the hypoxic tumor microenvironment, mouse splenocytes were cultured in 1% oxygen. Activation of iNOS was induced by LPS/IFN-γ and analysed by RT-PCR, Western blot and Griess assay. Radiosensitivity was analysed in a model of metabolic hypoxia by colony formation assay Results: The growth of both CT26 and 4T1 tumors in BALB/c mice was associated with an accumulation of MDSC in the spleen from 2-4% (in naive mice) to 8 and 35% respectively. Freshly isolated splenocytes and MDSC showed low ARG levels. Contrasting, hypoxia-conditioned splenocytes from tumor-bearing mice revealed a drastic transcriptional activation of ARG (up to 525.000-fold), and ARG+ MDSC. On the other hand splenocytes from tumor-free mice did not display ARG activation in hypoxic conditions. The iNOS/NO-mediated radiosensitization of mammary EMT6 tumor cells was strictly dependent on L-arginine levels, with a marginal effect of 1.2-fold at 10-30 μM while approaching a 2.4-fold effect at a physiological concentration of 125 μM. MDSC levels in CRC patients were increased, as compared with healthy donors (8-12% versus 4-5%). Patients with locally advanced CRC had 16-fold higher ARG expression in blood monocytes, as compared to healthy donors. 4 weeks after pre-operative radiotherapy the ARG levels decreased by 2-fold. Conclusions: Although tumor progression is clearly associated with the expansion of MDSC, their hypoxic conditioning appears to be crucial to uncover ARG as a tumor biomarker in vivo. iNOS/NO mediated radiosensitisation of hypoxic tumor cells was drastically impaired at low L-arginine levels which could be depleted by ARG+ MDSC. In a clinical setting, we consider ARG a potential biomarker in locally advanced rectal cancer. Citation Format: Wim Leonard, Inès Dufait, Heng Jiang, Femke Steenbeke, Marieke Vermeersch, Kalun Law, Guy Storme, Joeri Aerts, Valeri Verovski, Mark De Ridder. Myeloid-derived suppressor cells as a biomarker of tumor growth and radiosensitivity: Role of hypoxia-inducible arginase-1. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4986. doi:10.1158/1538-7445.AM2013-4986