Percentage Of Myeloid-derived Suppressor Cells Research Articles

Splenectomy suppresses growth and metastasis of hepatocellular carcinoma through decreasing myeloid-derived suppressor cells in vivo.

The function of the spleen in tumor development has been investigated for years. The relationship of the spleen with hepatocellular carcinoma (HCC), a huge health burden worldwide, however, remains unknown. The present study aimed to examine the effect of splenectomy on the development of HCC and the possible mechanism. Mouse hepatic carcinoma lines H22 and Hepa1-6 as well as BALB/c and C57 mice were used to establish orthotopic and metastatic mouse models of liver cancer. Mice were divided into four groups, including control group, splenectomy control group (S group), tumor group (T group) and tumor plus splenectomy group (T+S group). Tumor growth, metastases and overall survival were assessed at determined time points. Meanwhile, myeloid-derived suppressor cells (MDSCs) were isolated from the peripheral blood (PB), the spleen and liver tumors, and then measured by flow cytometery. It was found that liver cancer led to splenomegaly, and increased the percentage of MDSCs in the PB and spleen in the mouse models. Splenectomy inhibited the growth and progression of liver cancer and prolonged the overall survival time of orthotopic and metastatic models, which was accompanied by decreased proportion of MDSCs in the PB and tumors of liver cancer-bearing mouse. It was suggested that splenectomy could be considered an adjuvant therapy to treat liver cancer.

L-Arginine supplementation inhibits the growth of breast cancer by enhancing innate and adaptive immune responses mediated by suppression of MDSCs in vivo.

BackgroundL-Arg is involved in many biological activities, including the activation of T cells. In breast cancer patients, L-Arg is depleted by nitric oxide synthase 2 (NOS2) and arginase 1 (ARG-1) produced by myeloid-derived suppressor cells (MDSCs). Our aim was to test whether L-Arg supplementation could enhance antitumor immune response and improve survivorship in a rodent model of mammary tumor.MethodsTumor volumes in control and L-Arg treated 4 T1 tumor bearing (TB) BALB/c mice were measured and survival rates were recorded. The percentages of MDSCs, dendritic cells (DCs), regulatory T cells (Tregs), macrophages, CD4+ T cells, and CD8+ T cells were examined by flow cytometry. Additionally, levels of IL-10, TNF-α, and IFN-γ were measured by enzyme-linked immunosorbent assay (ELISA) and nitric oxide (NO) levels were measured by the Griess reaction. IFN-γ, T-bet, Granzyme B, ARG-1 and iNOS mRNA levels were examined by real-time RT-PCR.ResultsL-Arg treatment inhibited tumor growth and prolonged the survival time of 4 T1 TB mice. The frequency of MDSCs was significantly suppressed in L-Arg treated TB mice. In contrast, the numbers and function of macrophages, CD4+ T cells, and CD8+ T cells were significantly enhanced. The IFN-γ, TNF-α, NO levels in splenocytes supernatant, as well as iNOS, IFN-γ, Granzyme B mRNA levels in splenocytes and tumor blocks were significantly increased. The ARG-1 mRNA level in tumor blocks, the frequency of Tregs, and IL-10 level were not affected.ConclusionL-Arg supplementation significantly inhibited tumor growth and prolonged the survival time of 4 T1 TB mice, which was associated with the reduction of MDSCs, and enhanced innate and adaptive immune responses.

Identification of CD4+CD25+CD127- regulatory T cells and CD14+HLA-DR-/low myeloid-derived suppressor cells and their roles in the prognosis of breast cancer.

The aim of the present study was to identify cluster of differentiation 4+ (CD4+)CD25+CD127- regulatory T cells (Tregs) and CD14+ human leukocyte antigen-antigen D-related (HLA-DR-)/low myeloid-derived suppressor cells (MDSCs) in patients with breast cancer of varying stages, and investigate their roles and the potential interactions in the prognosis of breast cancer. A total of 40 patients with breast cancer were included in the study. A total of 30 healthy individuals served as the healthy control. Flow cytometry was performed for the identification of biomarkers. Natural Tregs were characterized by the expression of CD4+CD25+CD127-. The MDSC frequency was expressed as the percentage of CD33+CD11b+HLA-DR-lineage markers (Lin)-. The absolute number of Tregs was higher in breast cancer patients compared to the healthy control. The absolute number of Tregs in the patients with stage III or IV breast cancer was higher than those of the stage I or II, respectively. The percentage showed a gradual increase in the patients with breast cancer compared with the normal control. No direct correlation was established between the number or percentage of Tregs and the patient survival. There was a higher percentage of circulating MDSCs in breast cancer patients compared with the normal individuals. A close correlation was established between clinical cancer stage and percentage and total number of circulating MDSCs. To be exact, a significant increase of MDSC percentage and total number was observed in patients with stage III-IV breast cancer compared with the other cancer patients (stage I-II) and the normal individuals. No statistical difference was observed in the 3- and 5-year survival rates in the breast cancer patients with enhanced expression of Tregs, compared with the normal individuals. In conclusion, enhanced expression of CD4+CD25+CD127- Tregs cells and CD33+CD11+HLA-DR-LIN- MDSCs were identified from patients with breast cancer. Patients with advanced stage breast cancer showed upregulation of such cells. However, these 2 types of cells showed no correlation with the prognosis of breast cancer.

Dynamics of the spleen and its significance in a murine H22 orthotopic hepatoma model.

The dynamics of the spleen during tumor progression remains incompletely understood. In this study, we established a murine H22 orthotopic hepatoma model and dynamically detected alterations in the percentages of immunocytes in the spleen. We observed a prominent myeloid-derived suppressor cell (MDSC) accumulation during the early response which persisted through all the stages of tumor growth. In addition, the percentage of regulatory T cells (Tregs) increased by week 2. Although the percentage of CD3(+)CD49b(+) natural killer T (NKT) cells increased by day 3, and that of CD3(+)CD4(+) T cells slightly increased by week 1, they decreased to either normal or lower levels compared with those of normal mice. The percentages of total CD3(+), CD3(+)CD4(+), and CD3(+)CD8(+) T cells decreased by week 2, and that of NK cells decreased by week 3. The activation of non-Treg CD4(+) T cells was scarce. Moreover, splenic MDSCs of tumor-bearing mice suppressed the activation of splenocytes. Therefore, a negative immune response gradually prevailed over a positive immune response during tumor growth. In addition, splenectomy was performed at the time of tumor inoculation, and we found that splenectomy could prolong the survival time, reduce the tumor weights, decrease the ascites volumes, and ameliorate the immune status of the tumor-bearing mice. Splenectomy also decreased the percentage of MDSCs and increased the percentages of CD8(+) T cells, NK, and NKT cells in tumor tissues. Additionally, splenectomy decreased the percentage of MDSCs and increased that of CD8(+) T cells in peripheral blood. Overall, our findings suggest that immune-negative cells are dominant in the spleen during tumor progression. Splenectomy could be helpful to improve the immune responses of tumor-bearing hosts.

Toward harmonized phenotyping of human myeloid-derived suppressor cells by flow cytometry: results from an interim study.

There is an increasing interest for monitoring circulating myeloid-derived suppressor cells (MDSCs) in cancer patients, but there are also divergences in their phenotypic definition. To overcome this obstacle, the Cancer Immunoguiding Program under the umbrella of the Association of Cancer Immunotherapy is coordinating a proficiency panel program that aims at harmonizing MDSC phenotyping. After a consultation period, a two-stage approach was designed to harmonize MDSC phenotype. In the first step, an international consortium of 23 laboratories immunophenotyped 10 putative MDSC subsets on pretested, peripheral blood mononuclear cells of healthy donors to assess the level of concordance and define robust marker combinations for the identification of circulating MDSCs. At this stage, no mandatory requirements to standardize reagents or protocols were introduced. Data analysis revealed a small intra-laboratory, but very high inter-laboratory variance for all MDSC subsets, especially for the granulocytic subsets. In particular, the use of a dead-cell marker altered significantly the reported percentage of granulocytic MDSCs, confirming that these cells are especially sensitive to cryopreservation and/or thawing. Importantly, the gating strategy was heterogeneous and associated with high inter-center variance. Overall, our results document the high variability in MDSC phenotyping in the multicenter setting if no harmonization/standardization measures are applied. Although the observed variability depended on a number of identified parameters, the main parameter associated with variation was the gating strategy. Based on these findings, we propose further efforts to harmonize marker combinations and gating parameters to identify strategies for a robust enumeration of MDSC subsets.Electronic supplementary materialThe online version of this article (doi:10.1007/s00262-015-1782-5) contains supplementary material, which is available to authorized users.

Inflammaging-Associated Metabolic Alterations Foster Development of the MDS Genotype

Recent studies suggest that aging-associated inflammation, or "inflammaging", contributes to genetic instability and MDS predisposition. Although innumerable somatic genetic events have been annotated in recent years, including many that are not unique to MDS, they are not sufficient for disease initiation. The precise underlying mechanisms conducive to the emergence of these genetic events also remain to be delineated. We reported that bone marrow (BM) myeloid derived suppressor cells (MDSC) activated by the damage associated molecular pattern (DAMP) protein S100A9, promote ineffective hematopoiesis and the development of MDS. Inflammaging associated alterations in metabolism have been implicated in predisposition to cancer development with age. Here we report that S100A9 and ROS-induced inflammaging are associated with insulin resistance and hyperglycemia in the BM microenvironment that triggers activation of adaptive oncogenic pathways and genomic instability in HSPC. Glucose concentrations were markedly elevated in MDS BM plasma vs. age-matched controls, and directly correlated with S100A9 concentration (r=0.513, P=0.003, n=41). The magnitude of BM-glucose elevation significantly exceeded that in the peripheral blood and negatively correlated with the proportion of HSPC while directly correlating with BM MDSC percentage. S100A9 transgenic (Tg) mice displayed age-dependent elevation of glucose in peripheral blood and BM when compared to wild type mice accompanied by accumulation of somatic mutations (SM) by sequencing in aged S100A9-Tg compared to younger counterparts. NGS of 38 primary MDS BM specimens showed that SM common to MDS, such as those involving ASXL1, U2AF1 and DNMT3A, we re present only in high glucose stratified MDS-BM specimens (glucose > 110 ug/ml). Furthermore, there was a strong correlation between the cellular ROS/nuclear-β-catenin to DNA damage (γH2AX+ cells) linking S100A9-induced ROS accumulation to genetic instability. Elevation in BM plasma glucose was specifically associated with upregulation of the fat mass and obesity associated (FTO) transcript and protein in both MDS BM and S100A9Tg mice. FTO is a risk factor for type 2 diabetes, and encodes an α-ketoglutarate-dependent dioxygenase that functions as an RNA demethylase specific for N 6-methyladenosine (m6A) residues, targeted by splicing factors. Both human and murine MDS specimens displayed decreased m6A mRNA methylation compared to controls. FTO knockdown with CRISPR increased mRNA m6A methylation in MDS primary specimens, whereas overexpression of FTO led to a corresponding decrease that was enhanced by S100A9 stimulation. Moreover, S100A9 treatment induced FTO and demethylation of m6A mRNA in human and murine BM cells. These effects were accompanied by disruption of spliceosomes in the nucleus, as demonstrated by delocalization of SRSF2 from nuclear speckles into the cytoplasm where they colocalize with FTO in MDS patients. Interestingly, from the S100A9Tg mouse sequencing studies, we discovered that the mutation hotspots were in locations specific for histone H3K27 acetylation which have been previously linked to genomic instability, as well as splicing regulation, and regulated by histone deacetylase 1 (HDAC1). Reduced HDAC1 levels are known to make cells hypersensitive to DNA-damaging insults, such as those inducing ROS, similar to what we observed with treatment of S100A9 in healthy human bone marrow, MDS patient samples and S100A9Tg mice. Furthermore, this correlated with increased levels of DNA damage in our patient samples and in our murine S100A9Tg model as measured by phosphorylated γH2AX histones. Our studies provide a biological rationale for the initiation of DNA-genetic damage under inflammatory senescence conditions in MDS. These findings demonstrate that S100A9-induced inflammation activates a signaling cascade that enhances development of splicing variants and somatic gene mutations critical to MDS pathogenesis. DisclosuresList:Celgene Corporation: Honoraria, Research Funding.

Myeloid-derived suppressor cells enhance the expression of melanoma-associated antigen A4 in a Lewis lung cancer murine model.

The cancer-testis (CT) family of antigens are expressed in multiple types of malignant neoplasm and are silent in normal tissues, apart from the testis. Immunotherapy targeting CT antigens is a promising therapeutic strategy for treatment of solid tumors. One member of this family, melanoma-associated antigen A4 (MAGE-A4), has been demonstrated to be expressed in melanomas and lung cancer. Patients with tumors expressing the MAGE-A4 antigen exhibit specific cellular and humoral immune responses to the antigen, resulting in a favorable prognosis. Conversely, the expression of MAGE-A4 is associated with poor survival in lung cancer. Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of immunosuppressive cells, which are upregulated in the cancer microenvironment. Little is known regarding any potential correlation between the expression of MAGE-A4 antigens and the accumulation of MDSCs. The present study aimed to examine the association between circulating MDSC levels and MAGE-A4 expression in a mouse model of Lewis lung cancer. The expression of MAGE-A4 in tumor cells or tissues was evaluated using western blotting, while the percentage of MDSCs (CD11b+Gr-1+) in the blood was detected by flow cytometry. In addition, the suppressive capacity of MDSCs and the effectiveness of MDSC depletion were assessed in C57BL/6 tumor-bearing mice. MDSCs were demonstrated to upregulate MAGE-A4 expression via the phosphosphorylated-signal transducer and activator of transcription 3705 pathway, while depletion of MDSCs decreased the tumor growth rate, prolonged median survival and enhanced the recognition of MAGE-A4 by CD8+ T cells. These findings indicated that immunotherapeutic strategies involving induction of cytotoxic T lymphocytes that target MAGE-A4, in combination with MDSC depletion, may be an effective approach to immunotherapy for cancer types with high expression of MAGE-A4.

Relationship of preoperative and postoperative myeloid-derived suppressor cells percentage with the prognosis in rectal cancer patients

To observe the change of myeloid-derived suppressor cells (MDSCs) percentage in peripheral blood after operation in rectal cancer patients and to examine its association with the prognosis. Blood samples of pre-operation and postoperative 21-day from 64 stage I(-III( rectal cancer patients who underwent surgery in Department of General Surgery, The Affiliated Cancer Hospital, Zhengzhou University between January and December 2009 were collected. MDSCs percentage was detected by flow cytometry. Its association with the prognosis of patients was analyzed. MDSCs percentage of postoperative 21-day decreased significantly compared with pre-operation (P<0.01). When local recurrence or distant metastasis presented, MDSCs percentage increased again (all P<0.01) and reached the preoperative level(P>0.05). All the patients were further divided into two groups based on median MDSCs percentage. Patients with higher MDSCs percentage before operation (>3.78%) and after operation (>2.11%) had significantly lower 5-year overall survival(OS) (58.1% and 62.1%) and 5-year disease-free survival (DFS)(54.8% and 58.6%) as compared to those with lower MDSCs percentage(5-year OS 87.9% and 84.8%; 5-year DFS 82.8% and 80.0%, all P<0.05). Multivariate analysis showed that preoperative MDSCs percentage was an independent prognostic factor of rectal cancer(HR:4.065, 95% CI:1.026 to 16.108, P=0.04). Preoperative increased MDSCs percentage may be an important predictor of poor OS in rectal cancer patients. Dynamic change of MDSCs percentage can reflect the disease development.

IL-37b gene transfer enhances the therapeutic efficacy of mesenchumal stromal cells in DSS-induced colitis mice.

Aim:To investigate whether the transfer of the IL-37b gene, a newly identified inhibitor of both innate and adaptive immunity, could improve the therapeutic efficacy of mesenchumal stromal cells (MSCs) in inflammatory bowel disease (IBD).Methods:The expression of IL-37 in biopsied specimens of the patients with active ulcerative colitis (UC) was detected using RT-PCR and immunohistochemistry. Mice were treated with 3% dextran sulfate sodium (DSS) for 8 days to induce colitis. Before DSS treatment, the mice were injected with MSCs, MSC-eGFP or MSC-IL37b. Their body weight was measured each day, and the colons and spleens were harvested on d 10 for pathological and biochemical analyses.Results:In biopsied specimens of the patients with active UC, the expression of IL-37 was dramatically elevated in inflamed mucosa, mainly in epithelial cells and infiltrating immune cells. Compared to MSC-eGFP or MSCs, MSC-IL37b administration significantly attenuated the body weight and colon length reduction, and decreased the histological score in DSS-induced colitis mice. Furthermore, MSC-IL37b administration increased the percentage of myeloid-derived suppressor cells (MDSCs) among total splenic mononuclear cells as well as the percentage of regulatory T cells (Tregs) among splenic CD4+ T cells in the mice. Moreover, MSC-IL37b administration increased the IL-2+ cells and decreased the IFN-γ+ cells among splenic CD4+ T cells.Conclusion:IL-37 is involved in the pathophysiology of UC. IL-37b gene transfer enhances the therapeutic efficacy of MSCs in DSS-induced colitis mice by inducing Tregs and MDSCs and regulating cytokine production.

Study on the immunosuppressive activities of myeloid derived suppressor cells in patients suffering from recurrent spontaneous abortion

Objective To investigate the roles of myeloid derived suppressor cells (MDSCs) in the maintenance of maternal immune tolerance and to analyze their associations with recurrent spontaneous abortion (RSA). Methods Peripheral blood samples were collected from sixty subjects including non-pregnant healthy subjects, normal pregnant women and women with recurrent spontaneous abortion aged 25 to 35 years (n=20). Flow cytometry analysis was performed to monitor the changes of MDSCs at the 12th, 24th and 36th weeks of gestational age in three normal pregnant women. The differences with the numbers of MDSCs during the same stage of pregnancy in peripheral blood samples were compared among the three groups by flow cytometry analysis. RT-PCR analysis was used for detection of Arg-1 in women with normal pregnancy and RSA. CD8+ T cells were separated from non-pregnant healthy subjects by using magnetic beads. The immunosuppressive effects of MDSCs, isolated from women with normal pregnancy and RSA, on CD8+ T cells were evaluated by using the 3H thymidine incorporation assay. Results The number of MDSCs increased during normal pregnancy and the differences among different weeks were significant (12th week vs 24th week: P=0.0442, 24th week vs 36th week: P=0.0233, 12th week vs 36th week: P=0.0017). The percentages of MDSCs in peripheral blood of the healthy non-pregnant women were the lowest [(0.937±0.127)%], followed by those in women with RSA [(2.151±0.185)%] and normal pregnant women [(3.777±0.427)%]. There was significant difference with the percentage of MDSCs between women with normal pregnancy and RSA (P=0.0250). Compared with women with normal pregnancy, those with RSA showed significantly decreased levels of Arg-1 (P=0.0474) and suppressed activities of MDSCs against CD8+ T cell proliferation (P=0.0230). Conclusion The MDSCs might play an important role in the maintenance of maternal immune tolerance. Decreased number and inhibited function of MDSCs in the peripheral blood of women with RSA might be responsible for the development of RSA. Key words: Myeloid derived suppressor cell; Maternal immune tolerance; Recurrent spontaneous abortion

Effect of Cisplatin on the Frequency and Immuno-inhibitory Function of Myeloid-derived Suppressor Cells in A375 Melanoma Model.

To investigate the change of frequency and immuno-inhibitory function of myeloid-derived suppressor cells (MDSCs) after treatment of cisplatin (DDP) in A375 human melanoma model. BALB/c nude mice were inoculated with A375 cells to establish the human melanoma model and randomly divided into control group given normal saline (NS) and experimental group treated with DDP (5 mg/ kg). The percentages of MDSCs in the tumor tissue and peripheral blood after DDP treatment were detected by flow cytometry. The proliferation and interferon-γ (IFN-γ) secretion of T cells co-cultured with MDSCs were analyzed through carboxyfluorescein succinimidyl ester (CFSE) labeling assay and enzyme-linked immunospot (ELISPOT) assay, respectively. In A375 human melanoma model, DDP treatment could significantly decrease the percentage of MDSCs in the tumor tissue, but exerted no effect on the level of MDSCs in peripheral blood. Moreover, DDP treatment could attenuate the immuno-inhibitory function of MDSCs. T cells co-cultured with DDP-treated MDSCs could dramatically elevate the proliferation and production of INF-γ. DDP can decrease the frequency and attenuate immuno-inhibitory function of MDSCs in A375 melanoma model, suggesting a potential strategy to augment the efficacy of combined immunotherapy.

Impact of MDSC emergence during ovarian cancer in cytokine-deficient mice. (TUM6P.1011)

Abstract We have been studying the emergence of myeloid-derived suppressor cells (MDSCs) following i.p. transfer of ID8 cells (mouse ovarian surface epithelial cell carcinoma) into intact, syngeneic, C57BL/6J (wild type, WT) mice. Our current work studies the impact of the cytokine (CK) environment on ID8 expansion and MDSC emergence in the peritoneal cavity (PerC). We wanted to determine if particular pro- (IFNg) or anti- (IL4, IL10) inflammatory CKs were drivers of disease in this model. We found that, relative to WT mice, the development of hemorrhagic ascites was accelerated in IL10-/-, IL4-/-, and IFNgR-/- mice. The greatest percentage and number of MDSCs was observed in IL10-/- mice, which also had the greatest expansion of ID8 cells. ID8 triggered a marked increase in PerC Mfs, particularly in the IL10-/- mice, and lymphocytes, particularly in the IFNgR-/- and IL4-/- mice. However, for all mice, the percent representation of B cells in the total PerC cell pool declined markedly, particularly in the IL10-/- mice (&amp;gt; 70%). Interestingly, beyond the peritoneal locus of disease, both B and T lymphocyte percentages dropped in the spleens of all mice. This was seen without a concomitant increase in the number of MDSCs or appearance of ID8 in the spleen. These observations reinforce that inflammation drives MDSC emergence and suggest that changes in lymphocyte populations could serve as biomarkers for assessing ovarian cancer.

Clinical significance of plasmacytoid dendritic cells and myeloid-derived suppressor cells in melanoma

BackgroundImmune markers in the peripheral blood of melanoma patients could provide prognostic information. However, there is currently no consensus on which circulating cell types have more clinical impact. We therefore evaluated myeloid-derived suppressor cells (MDSC), dendritic cells (DC), cytotoxic T-cells and regulatory T-cells (Treg) in a series of blood samples of melanoma patients in different stages of disease.MethodsFlow cytometry was performed on peripheral blood mononuclear cells of 69 stage I to IV melanoma patients with a median follow-up of 39 months after diagnosis to measure the percentage of monocytic MDSCs (mMDSCs), polymorphonuclear MDSCs (pmnMDSCs), myeloid DCs (mDCs), plasmacytoid DCs (pDCs), cytotoxic T-cells and Tregs. We also assessed the expression of PD-L1 and CTLA-4 in cytotoxic T-cells and Tregs respectively. The impact of cell frequencies on prognosis was tested with multivariate Cox regression modelling.ResultsCirculating pDC levels were decreased in patients with advanced (P = 0.001) or active (P = 0.002) disease. Low pDC levels conferred an independent negative impact on overall (P = 0.025) and progression-free survival (P = 0.036). Even before relapse, a decrease in pDC levels was observed (P = 0.002, correlation coefficient 0.898). High levels of circulating MDSCs (>4.13%) have an independent negative prognostic impact on OS (P = 0.012). MDSC levels were associated with decreased CD3+ (P < 0.001) and CD3 + CD8+ (P = 0.017) T-cell levels. Conversely, patients with high MDSC levels had more PD-L1+ T-cells (P = 0.033) and more CTLA-4 expression by Tregs (P = 0.003). pDCs and MDSCs were inversely correlated (P = 0.004). The impact of pDC levels on prognosis and prediction of the presence of systemic disease was stronger than that of MDSC levels.ConclusionWe demonstrated that circulating pDC and MDSC levels are inversely correlated but have an independent prognostic value in melanoma patients. These cell types represent a single immunologic system and should be evaluated together. Both are key players in the immunological climate in melanoma patients, as they are correlated with circulating cytotoxic and regulatory T-cells. Circulating pDC and MDSC levels should be considered in future immunoprofiling efforts as they could impact disease management.

Pre- and post-operative evaluation: percentages of circulating myeloid-derived suppressor cells in rectal cancer patients.

This study aims to explore whether pre-operative or post-operative percentages of circulating myeloid-derived suppressor cells (MDSC) have any correlations with clinicopathological parameters in patients with rectal cancer and have any predictive values for local recurrence and disease free survival. Lin-/HLADR-/CD11b+/CD33+ MDSCs were analyzed in the study. Blood samples before and after surgery were measured by flow cytometry to determine the MDSC percentages. Plasma arginase Ilevels were analyzed using an enzyme-linked immunosorbent assay. Digital rectal examination, abdominal ultrasonography, or computed tomography was performed every three months after surgery to monitor local recurrence. Prior to surgery, cancer patients presented with higher levels of circulating MDSC compared to healthy controls (medians of 3.89% and 0.57%, respectively). Seven days after operation, we observed an immediate increase in MDSC followed by adowntrend. Local recurrence was observed in 11 of 41 patients (26.8%) with stage I/II cancer and 11 of 23 patients (47.8%) with stage III cancer. Significantly higher percentages of MDSC were observed in patients who had local recurrence versus patients without local recurrence. Multivariate analysis further verified that the pre-operative level of MDSC was significantly associated with local recurrence (P=0.038). Therefore, our results indicate that pre-operative percentage of MDSC appears to be areliable prognostic marker for local recurrence and poor disease free survival in rectal cancer patients with curative resection. myeloid-derived suppressor cells, rectal cancer, plasma arginase I, local recurrence, disease free survival.

Dendritic Cell Vaccination in Acute Leukemia Patients Induces Reduction of Myeloid-Derived Suppressor Cells : Immunological Analysis of a Pilot Study

Background and Purpose: Myeloid-derived suppressor cells (MDSC) are a heterogeneous cell population that have an immunesuppresive function and play an important role in tumor immunology and self tolerance. Elevated levels of MDSCs in peripheral blood (PB) are reported in various cancers and hematological malignancies such as MDS, NHL and MM. We reported that vaccination with Wilms’ tumor 1 (WT1) peptide-pulsed dendritic cells (DC) in NHL patients induced not only WT1 specific immune responses but also innate immunity which correlated with clinical efficacy (ASH 2013). In the present study, we focused on MDSCs and investigated the effects of DC vaccination on the alteration of MDSCs which have negative influence on disease progression.Patients and Methods: 8 patients with acute leukemia (6 AML and 2ALL patients; aged 16-77) who were treated with WT1 peptide-loaded DC vaccination were enrolled in the present study. MDSCs were analyzed by FACS. Linage negative, CD11b+CD33+HLA-DR- cells were defined as MDSC. Monocytic and granulocytic MDSC were defined as CD14+HLA-DRlow/- and CD15+CD11b+CD33+HLA-DR-, respectively. For proliferation assay, CD14+DR- cells and CD3+ T cells were isolated by magnetic beads method. CFSE-labeled CD3+ T cells were stimulated with anti-CD3 and anti-CD28 moAb together with IL-2 in the presence or absence of CD14+DR-cells for 4 days and cell division was analyzed by FACS. Cytoplasmic staining of indoleamine 2,3-deoxygenase (IDO) and arginase 1 were performed by permeabilization using saponin followed by moAb staining. Arginase 1 protein levels in plasma was assessed using a commercially available ELISA kit.Results: 3 patients were in complete remission (responder) and 5 others were in disease progression (non-responder) following one course of vaccination. WT1 specific T cell responses evaluated by a HLA-tetramer assay and a delayed type hypersensitivity (DTH) test were observed in all the responders and 2 non-responders. Mean percentage of MDSCs, monocytic and granulocytic MDSCs in PB was higher in acute leukemia patients than in healthy donors (1.60±0.72%, 16.56±6.07%, 1.34±0.69%, respectively in patients; 0.60±0.31%, 13.1±4.1%, 0.37±0.1%, respectively in healthy donors). Absolute number of circulating monocytic and granulocytic MDSCs before and after vaccination in responders was 359.4±117.8/μl and 35.2±14.8/μl, 282.8±119.2 /μl and 14.4±12.0/μl, respectively. On the other hand, those in non-responders were 126.3±60.2/μl and 36.3±21.7/μl, 181.3±124.0/μl and 65.1±12.0/μl, respectively. While DC vaccination resulted in reduction of both monocytic and granulocytic MDSCs by 21.3% and 59.1%, respectively in responders, it induced augmentation of these MDSCs by 43.5% and 79.3%, respectively in non-responders. A portion of MDSCs expressed IDO and arginase 1 in cytoplasm. While the percentage of IDO positive cells in monocytic MDSCs increased by 2.2 and 2.3 times in responders and non-responders, respectively following vaccination, the positivity of arginase 1 decreased by 59% in responders. On the other hand, the positivity of IDO and arginase 1 in granulocytic MDSCs decreased at the same level in both responders and non-responders following vaccination. CD14+DR-cells inhibited the proliferation of CD3+ T cells in a dose dependent manner, indicating that these cells share characteristics of MDSCs. The inhibition of the proliferation of CD3+T cells was partially restored by the addition of 1-methyl-DL-tryptophan (IDO inhibitor) and Nω-hydroxy-nor-arginine (arginase 1 inhibitor), suggesting that the inhibition was mediated at least in part by IDO and arginase 1. Amount of arginase 1 in the plasma before and after vaccination decreased by 25% in responders, not in non-responders.Conclusions: These results demonstrated that DC vaccination resulted in the reduction of the absolute number of MDSCs which seems to be correlated with clinical efficacy. DC vaccination may have inhibitory effects on several functions of MDSCs that can suppress immune surveillance against leukemia, which is favorable for regression of leukemia cells. DisclosuresNo relevant conflicts of interest to declare.

Myeloid-derived suppressor cells regulate immune response in patients with chronic hepatitis B virus infection through PD-1-induced IL-10.

Although myeloid-derived suppressor cells (MDSCs) are well known for their immunosuppressive function in several pathological conditions, the role of MDSCs in hepatitis B virus infection remains obscure. In this study, we investigated the frequency and function of MDSCs in the peripheral blood and liver of 91 chronic hepatitis B (CHB) patients. A higher percentage of MDSCs, defined as CD14(+)HLA-DR(-/low), was detected in peripheral blood of CHB patients than that of the healthy controls. Moreover, high expression of programmed death 1 (PD-1) and secretion of IL-10 in this population were determined. The frequency of MDSCs was positively correlated with serum viral load, but it was negatively correlated with liver inflammatory injury. These cells were also abundant in liver tissue of CHB patients and were related to necroinflammatory activity. Furthermore, we found that these cells could suppress hepatitis B virus-specific CD8(+) T cell response, including reduced proliferation and IFN-γ production, and inhibit degranulation of CD8(+) T cells, including reduced production of granzyme B and perforin. Importantly, PD-1-induced IL-10 production by MDSCs was responsible for the suppressive activity. To our knowledge, for the first time our study proved that CD14(+)HLA-DR(-/low)PD-1(+) MDSCs in CHB patients contribute to an inadequate immune response against the virus and lead to chronic infection, which represents a potential target for therapeutic intervention.

Increased monocytic CD14⁺HLADRlow/- myeloid-derived suppressor cells in obesity.

Obesity is associated with numerous immunological disorders. The present study investigated the proportion and phenotype of myeloid‑derived suppressor cells (MDSCs) in the plasma of obese subjects and the association of these cells with the level of liver enzymes. Certain features of the immune response in obese subjects were examined by analyzing the expression of T cell receptor‑ζ (TCRζ) molecules on the surface of T cells. The expression and secretion of S100A9, a possible marker for MDSCs, were detected in the peripheral blood of obese subjects and compared with levels in lean controls. Results showed that the percentage of monocytic MDSCs, with the phenotype CD33+CD11b+CD14+HLADRlow/‑, was significantly increased in obese subjects compared with lean controls. The circulating level of monocytic MDSCs was positively correlated with the levels of liver enzymes in serum. The expression of the TCRζ molecule in the resting T cells was significantly lower in obese individuals than that of lean controls. The expression of S100A9 was detected in the majority of monocytes in peripheral blood mononulear cells, but no difference was identified in the frequency of CD14+S100A9+ cells between the obese and lean groups. However, the plasma level of S100A8/9 was significantly increased in obese compared with lean subjects. These observations suggested that the increased frequency of CD33+CD11b+CD14+HLADRlow/‑ cells may be responsible for the impaired T‑cell function and liver injury observed in obesity.

Cyclophosphamide and IL-12-transduced DCs enhance the antitumor activity of tumor antigen-stimulated DCs and reduce Tregs and MDSCs number.

A hostile tumor microenvironment, characterized by an abundance of T regulatory cells and myeloid-derived suppressor cells (MDSCs), considerably limits the efficacy of dendritic cell (DC)-based vaccines. The intention of this study was to enhance the antitumor activity of vaccines consisting of bone marrow-derived DCs stimulated with TAg (BMDC/TAg) via single administration of cyclophosphamide and multiple injections of interleukin (IL)-12-transduced DCs (BMDC/IL-12). The combined chemoimmunotherapy was applied in the treatment of mice with subcutaneously (SC) growing, advanced MC38 colon carcinoma. The highest level of tumor growth inhibition, accompanied by high cytotoxic activity of effector cells, and their increased influx into tumor tissue, was observed after application of cyclophosphamide in combination with BMDC/TAg and BMDC/IL-12. The effect was probably associated with the elimination of T regulatory cells from spleens and tumors, but most of all with changes in the number and differentiation stage of MDSCs. After the therapy, the percentage of granulocytic and monocytic MDSCs in spleens was significantly lower than in the control group. Moreover, MDSCs derived from spleens and tumors showed increased expression of MHC class II, which may indicate the higher maturation stage of the myeloid cells as well as their enhanced capacity toward antigen presentation. The obtained data indicate that the optimal composition of antitumor vaccines able to limit the suppressor activity of MDSCs is essential to enhance the elimination of tumor cells and to achieve an optimal therapeutic effect.

PM-16 * IMMUNOSUPPRESSIVE FACTORS, MDSC AND ARGINASE, ARE ELEVATED IN DOGS WITH MALIGNANT GLIOMA

Immunotherapy for malignant gliomas has striking efficacy in rodent models but very limited effects in clinical trials, which may reflect immunological differences between induced and spontaneous tumors. The monocytes that heavily infiltrate GBM develop myeloid-derived suppressor cell (MDSC) phenotype, and these immunosuppressive cells are increased in GBM patients, but not in murine models. Canine primary brain tumors may offer a more faithful representation of the human disease due to similarities in clinical and pathobiological characteristics. We were recently first to identify MDSC in dogs and found significantly increased in number in dogs with advanced cancers. To add further support to pet dogs with glioma as a translational model, we assessed whether canine glioma patients also have elevated numbers of MDSC relative to healthy dogs. MDSC secrete arginase that removes L-arginine from the microenvironment profoundly impairing T cell proliferation and function. Therefore we also measured serum levels of arginase 1 in all dogs. The mean percentage of monocytic MDSC in peripheral blood of dogs with glioma (n = 26) was 7.51 ± 1.31, which was significantly greater than healthy controls (n = 10) with 0.74 ± 0.34 (P< 0.0001). The median percentages of granulocytic MDSC did not differ between dogs with glioma versus controls, 15.95% and 12.5% respectively. There were also significantly increased levels of arginase in the serum of dogs with gliomas, 8.00 ± 3.32 U/L, relative to that of healthy controls, 2.40 ± 1.40 U/L (P < 0.0001). These data demonstrate that some of the immunosuppressive mechanisms present in human gliomas patients are also found in dogs with malignant gliomas. These findings lend further support that the pet dog with spontaneous brain cancer that shares our environment is an excellent translational model for the human disease. We are currently working to correlate changes in numbers of MDSC and arginase levels in serum with disease burden as possible biomarkers for response to therapy.

Abstract 1099: Sorafenib increases numbers and functions of tumor-infiltrated T cells and enhances therapeutic outcomes of adoptive T cell therapy by modifying tumor microenvironment

Abstract Purpose: Many chemotherapeutic agents are known to have immunomodulatory activity to improve the effectiveness of adoptive T-cell therapy. We hypothesized that sorafenib could reverse the immunosuppression of tumor microenvironment to enhance T-cell mediated tumor regression. Experimental design: OVA-specific CD8+ T cells were co-cultured with sorafenib-pretreated OVA-overexpressing E.G7 mouse lymphoma cells to evaluate their function using flow cytometry, 51Cr-release assay, and transwell assay. The underlying mechanism of sorafenib-mediated immunomodulation was also explored by Western blotting. In animal studies, the activation of OVA-specific CD8+ T cells transduced with a granzyme B promoter-driven imaging reporter was monitored by bioluminescence imaging and the levels of regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSCs) were assessed by flow cytometry. Results: We found that sorafenib suppressed the expression of critical immunosuppressive components, including TGF-β , IL-10, VEGF, indoleamine 2,3-dioxygenase (IDO) and CCL2/MCP-1, in E.G7 cells through the inhibition of STAT3 signaling pathway and decreased the percentage of Tregs and MDSCs in tumors. Surprisingly, the anti-tumor effect of small numbers of CD8+ T cells was substantially enhanced when combined with sorafenib in vivo. Citation Format: Jeng-Jong Hwang, Hui-Yen Chuang, Ya-Fang Chang. Sorafenib increases numbers and functions of tumor-infiltrated T cells and enhances therapeutic outcomes of adoptive T cell therapy by modifying tumor microenvironment. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1099. doi:10.1158/1538-7445.AM2014-1099