The Journal of International Medical Research 2011; 39:

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1 The Journal of International Medical Research 211; 39: Increased Circulating Immunosuppressive CD14 + HLA-DR /low Cells Correlate with Clinical Cancer Stage and Pathological Grade in Patients with Bladder Carcinoma X-K YUAN 1, X-K ZHAO 2, Y-C XIA 1, X ZHU 2 AND P XIAO 1 1 Department of Nephrology, and 2 Department of Urology, Second Xiangya Hospital, Central South University, Changsha, China This study investigated CD14 + HLA-DR /low cells in peripheral blood mononuclear cells (PBMCs) from 64 patients with bladder carcinoma (BC) and 14 healthy controls. Cell phenotypes were determined and CD14 + HLA-DR /low cells, CD14 + HLA- DR + cells and PBMCs depleted of CD14 + HLA-DR /low cells were isolated. Proliferation of stimulated PBMCs and interferon-g (IFN-g) production after addition of CD14 + HLA-DR /low and CD14 + HLA-DR + cells at different ratios were measured. IFN-g production was also measured after addition of L-arginine and/or antitransforming growth factor-b (TGF-b) neutralizing monoclonal antibody, and in PBMCs depleted of CD14 + HLA-DR /low cells. The proportion of CD14 + HLA-DR /low cells in BC patients was significantly higher than in controls. CD14 + HLA-DR /low cells significantly decreased T-cell proliferation and IFN-g production in a dose-dependent manner. This suppressive activity was partially reversed by L-arginine or anti-tgf-b. Enhanced IFN-g secretion was also seen in PBMCs depleted of CD14 + HLA-DR /low cells. The level of CD14 + HLA-DR /low cells was associated with gender, tumour size, number of tumours, cancer pathological grade and clinical stage. CD14 + HLA- DR /low cells may represent a subpopulation of myeloid-derived suppressor cells in BC patients. KEY WORDS: BLADDER CARCINOMA; MYELOID-DERIVED SUPPRESSOR CELLS; TUMOUR EVASION Introduction Bladder carcinoma (BC) is the second most common urological neoplasm. 1 Around 8% of newly diagnosed bladder cancers are staged as non-muscle-invasive BC. Bacillus Calmette Guérin (BCG) immunotherapy is currently the standard treatment for preventing local recurrence and tumour progression after transurethral resection of high-risk non-muscle-infiltrating BC. A substantial proportion of BCG-treated carcinomas recur within 5 years. 2,3 This may 1381

2 be partially explained by a state of immune anergy/tolerance, which is a well-described phenomenon in cancer patients. 4 Recent studies have reported that myeloid-derived suppressor cells (MDSCs) are one of the key types of suppressor cell that regulate antitumour immune responses in tumour-bearing animals and cancer patients. 5,6 MDSCs suppress T-cell function by a number of mechanisms, including a high level of arginase activity as well as nitric oxide and reactive oxygen species production. 5 9 In mice, MDSCs were originally described as a population of CD11b + Gr1 + cells. 5 More recently, MDSCs were divided into two subsets based on the presence of the molecules, Ly6C and Ly6G: cells with a granulocytic phenotype, positive for the Ly6G marker; and cells with a monocytic phenotype, positive for the Ly6C marker. 1,11 In contrast to murine models, MDSCs in humans are less well characterized and no specific markers have been identified. In cancer patients, MDSCs are most commonly defined as cells that are positive for the common myeloid marker CD33, but lack markers of mature myeloid and lymphoid cells and of the major histocompatibility complex class II molecule human leucocyte antigen (HLA)-DR. 12,13 A CD15 + population with MDSC characteristics has, however, been described in renal cell carcinoma. 14,15 More recently, studies have identified the existence of a monocytic subset of MDSCs with the phenotype CD14 + HLA-DR /low in patients with various types of cancer These findings suggest that MDSCs in cancer patients are a group of phenotypically heterogeneous cells that have only their immunosuppressive activities in common. The present study investigated the phenotypic and functional features of MDSCs in BC patients. Patients and methods PATIENTS Consecutive patients with newly diagnosed or recurrent BC with a histological diagnosis of transitional cell carcinoma, seen and treated at the Second Xiangya Hospital, Changsha, China, between January 28 and December 29, were enrolled in the study. All bladder tumours were classified using TNM (tumour, node, metastasis) 22 and World Health Organization 1973 classifications. 22,23 Patients with recurrent BC had not received any treatment for at least 8 weeks. Healthy controls matched for age and gender to the patient population attending the clinic of the Second Xiangya Hospital for routine examination were also recruited over the same period. BC patients with any inflammatory or autoimmune diseases were excluded from the study. Peripheral blood was collected from the BC patients preoperatively, prior to administering any radiation or chemotherapy. In patients who specifically consented, peripheral blood was also collected 1 week postoperatively. Peripheral blood was collected from healthy controls in the morning, when they came for routine examination. Written informed consent for the main study was obtained from all the study participants. The study was conducted in accordance with the Declaration of Helsinki and the study protocol was approved by the Ethics Committee of the Second Xiangya Hospital, Central South University. ANTIBODY STAINING AND ANALYSIS Peripheral blood mononuclear cells (PBMCs) from BC patients and healthy controls were isolated using Ficoll density gradient centrifugation. To determine the frequency of Lin HLA-DR CD33 + cells, CD14 + HLA-DR /low 1382

3 cells and CD4 + CD25 + CD127 regulatory T (T reg ) cells, multicolour fluorescence-activated cell sorting analysis was performed using the following antibodies: phycoerythrin cyanine 5 (PC5)-conjugated anti-cd33 (BD Biosciences, Franklin Lakes, NJ, USA); phycoerythrin (PE)- conjugated anti-cd3, -CD14, -CD19 and -CD56 and fluorescein isothiocyanate (FITC)- conjugated anti-hla-dr (Immunotech, Westbrook, ME, USA); PC5-conjugated anti- CD4, PE-conjugated anti-cd25 and FITCconjugated anti-cd127 (all from ebioscience, San Diego, CA, USA). Isotype-matched antibodies were used as controls for all samples. Cell samples were incubated with the fluorescent conjugated primary antibodies for 2 min on ice at the dilution suggested by the manufacturer. Flow cytometry was performed using an EPICS ELITE ESP flow cytometer equipped with EXPO 32 software (Beckman Coulter, Brea, CA, USA). FLUORESENCE ACTIVATED CELL SORTING The CD14 + HLA-DR /low and CD14 + HLA-DR + cells were sorted from the PBMCs using the BD FACSVantage SE cell sorting system (BD Biosciences). PBMCs depleted of CD14 + HLA- DR /low cells were also sorted. The purity of the cells after sorting was > 95%. PROLIFERATION ASSAYS A total of 1 4 PBMCs from BC patients and normal controls were stimulated with 1 µg/ml phytohaemagglutinin (PHA) (Sigma- Aldrich, St Louis, MO, USA) plus 5 ng/ml phorbol 12-myristate 13-acetate (PMA) (Sigma-Aldrich) and cultured for 3 days at 37 C. The PBMCs were then cocultured with autologous sorted CD14 + HLA-DR /low or CD14 + HLA-DR + cells at different ratios. Proliferation was measured after 3 days by 3 H-thymidine incorporation (Amersham Biosciences, Piscataway, NJ, USA). Interferon-γ (IFN-γ) concentration was determined in the supernatants after 2 days of culture using an enzyme-linked immunosorbent assay (ELISA) (ebioscience) according to the manufacturer s instructions. In addition, IFN-γ concentration was measured in PBMCs from BC patients cultured with autologous CD14 + HLA-DR /low cells after the addition of 5 µmol/l L- arginine and/or 1 µg/ml anti-transforming growth factor-β (TGF-β) blocking monoclonal antibody (mab) (R&D Systems, Minneapolis, MN, USA) for 3 days at 37 C. The IFN-γ concentration was also measured in PBMCs from BC patients depleted of CD14 + HLA- DR /low cells following stimulation with PHA 1 µg/ml plus PMA 5 ng/ml and culture for 3 days at 37 C. MEASUREMENT OF ARGINASE ACTIVITY Arginase activity was measured in CD14 + HLA-DR /low and CD14 + HLA-DR + cells from BC patients and normal controls after the addition of L-arginine. Arginase was quantified by measuring the production of urea, as described by Sinha et al. 24 The urea concentration was measured at 54 nm using a 96-well microplate reader. MEASUREMENT OF TGF-b SECRETION The ability of CD14 + HLA-DR /low and CD14 + HLA-DR + cells from BC patients to secrete TGF-β was measured by adding 1 µg/ml anti-tgf-β 1 blocking mab to wells containing 1 5 cells. After 12 h, the cultures were tested for total TGF-β 1 using an ELISA kit (ebioscience), according to the manufacturer s instructions. STATISTICAL ANALYSES Data are presented as the mean ± SD. All 1383

4 analyses were performed using SPSS software package, version 13. (SPSS Inc., Chicago, IL, USA) for Windows. Pearson s correlation coefficient was used to assess the relationship between CD14 + HLA-DR /low cells and CD4 + CD25 + CD127 cells. A Student s t- test was used to analyse differences between groups. A P-value <.5 was considered to be statistically significant. Results PATIENTS A total of 64 patients with BC were enrolled in the study: 48 (75%) were male and 16 (25%) were female. The median patient age was 68 years (range years); 41 (64%) had newly diagnosed BC and 23 (36%) had recurrent BC. In addition, 14 healthy controls were recruited to the study: 1 (71.43%) were male and four (28.57%) were female. The median age for control subjects was 65 years (range years). CELL PHENOTYPE ANALYSIS Similar low levels of Lin HLA-DR CD33 + cells were detected in BC patients and healthy A 1 3 BC patient Normal control No. of HLA-DR cells No. of CD14 cells No. of CD14 cells B 3 CD14 + HLA-DR /low cells (%) BC patients Normal controls FIGURE 1: (A) Representative flow cytometry plots showing decreased human leucocyte antigen (HLA)-DR in peripheral blood mononuclear cells (PBMCs) from a patient with bladder carcinoma (BC) and a normal control. (B) CD14 + HLA-DR /low cell frequencies were measured using flow cytometry in PBMCs from individual BC patients (n = 64) and controls (n = 14) (horizontal black line shows the mean; P <.1 versus normal controls, Student s t-test) 1384

5 controls (mean ± SD 1.35% ±.87% versus 1.22% ±.43%). In contrast, the proportion of CD14 + HLA-DR /low cells in BC patients was significantly higher than in healthy controls (P <.1) (Fig. 1); low or undetectable HLA- DR was seen in 49% (range 17 8%) of circulating CD14 + monocytes in BC patients, whereas only 32% (range 14 54%) had this phenotype in healthy controls. There was no difference in the overall frequency of CD14 + monocytes in peripheral blood between BC patients and healthy controls (data not shown). The proportion of CD4 + CD25 + CD127 T reg cells in BC patients was significantly higher than in healthy controls (mean ± SD 3.82% ± 2.4% versus 1.13% ±.67%; P <.1), and the level was not significantly correlated with levels of CD14 + HLA-DR /low cells (Fig. 2). ANALYSIS OF SUPPRESSIVE ACTIVITY CD14 + HLA-DR /low cells significantly decreased IFN-γ production and T-cell proliferation in a dose-dependent manner (P <.1) (Fig. 3). In contrast, CD14 + HLA- DR + cells, even at the highest ratio (4 : 1), were not suppressive (data not shown). Arginase activity was measured in CD14 + HLA-DR /low and CD14 + HLA-DR + cells from BC patients and controls. As shown in Fig. 4A, arginase activity in CD14 + HLA- DR /low cells from BC patients was more than two-fold higher (mean ± SD ± 15.3 µmol urea/1 5 cells) than in CD14 + HLA-DR + cells (mean ± SD 14.7 ± 9.5 µmol urea/1 5 cells) and CD14 + HLA-DR /low and CD14 + HLA-DR + cells from normal controls (P <.1 for all comparisons). In contrast, no difference in arginase activity between CD14 + HLA-DR /low and CD14 + HLA-DR + cells was seen in healthy controls (mean ± SD, 96.1 ± 14.9 and 95.4 ± 12.8 µmol urea/1 5 cells, respectively). The ability to secrete TGF-β ex vivo was measured in CD14 + HLA-DR /low and CD14 + HLA-DR + cells from BC patients. The TGF-β level from CD14 + HLA-DR /low cells was.7 ng/1 5 cells, whereas CD14 + HLA-DR + cells did not produce detectable levels of this cytokine. The addition of L-arginine or anti-tgf-β 1 12 CD4 + CD25 + CD127 /low T reg cells (%) CD14 + HLA-DR /low cells (%) FIGURE 2: Scattergram showing lack of correlation between CD14 + HLA-DR /low cells and CD4 + CD25 + CD127 /low regulatory T (T reg ) cells in peripheral blood mononuclear cells from patients with bladder carcinoma (n = 64, r =.137, not statistically significant, P >.5) 1385

6 A 4 B 4 IFN-γ (pg/ml) T cells (counts/min 1) : 1 2 : 1 1 : 1 : 1 4 : 1 2 : 1 1 : 1 : 1 CD14 + HLA-DR /low : PBMC CD14 + HLA-DR /low : PBMC FIGURE 3: (A) Interferon-γ (IFN-γ) production and (B) T-cell proliferation, measured using 3 H-thymidine incorporation into peripheral blood mononuclear cells (PBMCs), from patients with bladder carcinoma after the addition of purified autologous CD14 + HLA-DR /low cells at different ratios (mean ± SD of three independent experiments; P <.1, Student s t-test) blocking mab to PBMC cultures partially reversed the suppressive activity of CD14 + HLA-DR /low cells (Fig. 4B). Furthermore, the addition of both L-arginine and anti-tgf-β 1 blocking mab almost completely reversed the suppression of IFN-γ production by CD14 + HLA-DR /low cells (Fig. 4B). Enhanced IFN-γ secretion was seen in PBMCs depleted of CD14 + HLA-DR /low cells (Fig. 4B). A Urea (µmol per 1 5 cells) CD14 + HLA-DR + CD14 + HLA-DR /low BC patients CD14 + HLA-DR + CD14 + HLA-DR /low Normal controls B IFN-γ (pg/ml) PBMC * PBMC + L-arginine PBMC + L-arginine + anti-tgf-β 1 PBMC + anti-tgf-β 1 PBMC depleted CD14 + HLA-DR /low cells FIGURE 4: (A) Arginase activity quantified using the production of urea in CD14 + HLA- DR /low and CD14 + HLA-DR + cells from normal controls and patients with bladder carcinoma (BC). (B) Interferon-γ (IFN-γ) production by peripheral blood mononuclear cells (PBMCs) after the addition of CD14 + HLA-DR /low cells alone, in the presence of L- arginine and/or antitransforming growth factor-β 1 (anti-tgf-β 1 ) monoclonal antibody, and in PBMCs depleted of CD14 + HLA-DR /low cells (mean ± SD of three independent experiments; *P <.5, P <.1, Student s t-test) 1386

7 PRE- AND POSTOPERATIVE LEVELS OF CD14 + HLA-DR /low CELLS Postoperative blood samples were available for 16 patients. Compared with preoperative values, the frequency of CD14 + HLA-DR /low cells was significantly decreased 1 week after surgery (P <.1) (Fig. 5). CD14 + HLA-DR /low CELLS AND CLINICOPATHOLOGICAL FACTORS Correlations between the level of CD14 + HLA- DR /low cells and various clinicopathological factors are given in Table 1. The CD14 + HLA- DR /low level was not related to age or tumour recurrence, but was associated with gender, tumour size and number of tumours (P <.5). Furthermore, significant associations between the level of CD14 + HLA-DR /low cells and cancer pathological grade and clinical stage were also observed (both P <.1). Discussion Bacillus Calmette Guérin is commonly used as the first-line adjuvant treatment for nonmuscle-invasive BC following transurethral resection. A substantial proportion of BCGtreated patients experience BC recurrence within 5 years, however, 2,3 and the mechanism of resistance to BCG has yet to be elucidated. One factor may be tumourinduced immune suppression, a condition that accompanies tumour progression and occurs in many patients with advanced cancer. 4,25 Various populations of cells shown to mediate immune suppression have been identified in cancer patients. Among them, MDSCs have become the focus of intense study. The accumulation of myeloid cells that suppress T-cell activation in tumourbearing mice was first reported in the late 198s, 26,27 but their functional importance in the immune system has only been acknowledged more recently. To date, accumulating evidence has shown that MDSCs are a largely heterogeneous group of immature myeloid cells composed of macrocytic/monocytic, granulocytic or dendritic precursor cells or myeloid cells in the early stages of differentiation. 5 These cells are well characterized in mice as having a CD11b + Gr1 + phenotype, but the absence of 14 CD14 + HLA-DR /low cells (%) Preoperation Postoperation FIGURE 5: Percentages of CD14 + HLA-DR /low cells in peripheral blood mononuclear cells from individual patients with bladder carcinoma preoperation and 1 week post - operation (n = 16; horizontal black line shows the mean; P <.1, Student s t-test) 1387

8 TABLE 1: Correlations between the frequency of CD14 + HLA-DR /low cells and clinicopathological factors in patients with bladder carcinoma No. of CD14 + HLA-DR -/low Statistical Clinicopathological factors patients cells (%) significance a Age NS < 65 years ± years ± Gender P =.367 Male ± 4.45 Female ± 3.19 Tumour size P =.314 < 3 cm ± cm ± 3.54 No. of tumours P =.429 Single ± Multiple ± 4.48 Clinical stage P =.18 Tis/Ta/T ± T2 T ± Tumour grade P <.1 G1/G ± G ± 4.29 Recurrent tumour NS Yes ± No ± Data presented as mean ± SD. a Student s t-test was used to analyse differences between groups. All bladder tumours were classified using TNM (tumour, node, metastasis) 22 and World Health Organization 1973 classifications. NS, not statistically significant (P >.5). specific markers on MDSCs in humans means that only limited information is available. In agreement with recent reports, the present study demonstrated significantly increased numbers of CD14 + HLA-DR /low cells in the peripheral blood of patients with BC compared with healthy controls. In the present study, again consistent with other reports, 16,17,19 there was no difference in the proportion of Lin HLA-DR CD33 + cells between BC patients and healthy controls: this phenotype has frequently been described in connection with MDSCs in cancer patients. The CD14 + HLA-DR /low cells have been identified in various human malignancies A number of different mechanisms by which these cells suppress T- cell function have been reported in patients with different cancer histotypes. Filipazzi et al. 16 found that the suppressive activity of CD14 + HLA-DR /low cells in the peripheral blood of melanoma patients was mediated by TGF-β, whereas no involvement of the arginase or inducible nitric oxide synthase pathways was detected. In contrast, another study showed that the suppression of T-cell proliferation by circulating CD14 + HLA- DR /low cells from melanoma patients was dependent on arginine 1 and oxidative 1388

9 stress, and required cell contact. 17 Hoechst et al. 18 demonstrated that suppression by CD14 + HLA-DR /low cells in hepatocellular carcinoma patients was mediated through arginase activity and the induction of T reg cells. In a second study, Hoechst et al. 28 also showed that CD14 + HLA-DR /low cells inhibited autologous natural killer (NK) cell cytotoxicity and cytokine release in patients with hepatocellular carcinoma when cocultured in vitro. Blocking of arginase function did not, however, affect the inhibitory effect of CD14 + HLA-DR /low cells, and CD14 + HLA-DR /low cell-mediated inhibition of NK cell function was shown to be mainly dependent on the NKp3 receptor on NK cells. 28 In the present study, CD14 + HLA-DR /low cells in the peripheral blood of BC patients displayed clear suppressive activity on T cells and further investigation revealed that this suppression mainly involved TGF-β and arginase activity. Taken together, these findings suggest that CD14 + HLA-DR /low cells in cancer patients are diverse and can differ in function, probably depending on factors secreted by the tumour. Nevertheless, as suppressive activity is the gold standard of MDSCs, CD14 + HLA-DR /low cells in BC patients can be viewed as being MDSCs. 29 Based on findings demonstrating a potential induction of T reg cells by MDSCs, 18,3,31 the relationship between CD14 + HLA-DR /low cells and CD4 + CD25 + CD127 /low T reg cells in BC patients was investigated. 32,33 In mice, it was shown that MDSCs indirectly suppressed T- cell activation by inducing T reg cells which, in turn, downregulated cell-mediated immunity. 9,31 More recently, Hoechst et al. 18 reported that CD14 + HLA-DR /low cells from hepatocellular carcinoma patients induced T reg cells in vitro. A correlation between circulating CD14 + HLA-DR /low cells and CD4 + CD25 + CD127 /low T reg cells was not, however, seen in the present study. This is consistent with a recent report showing that the percentage of T reg cells was high throughout tumour growth and did not relate to the kinetics of expansion of the MDSC population, suggesting that MDSCs are not involved in T reg cell induction. 11 The accumulation of MDSCs is regulated by many factors, mostly released by tumour cells, which promote expansion of the MDSCs population through the stimulation of myelopoiesis and inhibition of the differentiation of mature myeloid cells. 5 In addition, other factors produced mainly by activated T-cells and tumour stromal cells are involved in directly activating MDSCs. 5 CD14 + HLA-DR /low cells are most likely to be a peripheral MDSC population, which is induced mainly by tumour-secreted factors. The dramatic decrease in the proportion of circulating CD14 + HLA-DR /low cells seen after tumour resection is consistent with this hypothesis. Investigation of associations between CD14 + HLA-DR /low cells and clinicopatho - logical characteristics in BC patients in the present study revealed that the proportion of CD14 + HLA-DR /low cells did not correlate with patient age or tumour recurrence, but was associated with gender, tumour size and number of tumours. More importantly, significant associations between CD14 + HLA- DR /low cells and cancer pathological grade and clinical stage were also observed. These findings strongly indicate that CD14 + HLA- DR /low cells are associated with disease progression and are clinically important mediators of tumour-mediated immune suppression in patients with BC. In conclusion, an increase in the frequency of immunosuppressive MDSCs, characterized by their CD14 + HLA-DR /low phenotype, was seen in the peripheral blood 1389

10 of patients with BC. CD14 + HLA-DR /low cells displayed strong T-cell suppressive activity, which mainly involved arginase and TGF-β production. Moreover, there was a significant correlation between circulating CD14 + HLA-DR /low cells and clinical cancer stage and pathological grade. On the basis of these findings, CD14 + HLA-DR /low cells may represent a subpopulation of MDSCs in BC patients. Effective MDSC elimination will be critical for successful immunotherapy, not only in patients with BC but also in patients with other types of cancer. Further investigations are required to assess the immunosuppressive function of MDSCs in solid malignancies, including BC. Conflicts of interest The authors had no conflicts of interest to declare in relation to this article. Received for publication 16 March 211 Accepted subject to revision 23 March 211 Revised accepted 8 July 211 Copyright 211 Field House Publishing LLP References 1 Jemal A, Siegel R, Xu J, et al: Cancer statistics, 21. CA Cancer J Clin 21; 6: Järvinen R, Kaasinen E, Sankila A, et al for the FinnBladder Group: Long-term efficacy of maintenance bacillus Calmette Guérin versus maintenance mitomycin C instillation therapy in frequently recurrent TaT1 tumours without carcinoma in situ: a subgroup analysis of the prospective, randomised FinnBladder I study with a 2-year follow-up. Eur Urol 29; 56: Malmström PU, Sylvester RJ, Crawford DE, et al: An individual patient data meta-analysis of the long-term outcome of randomised studies comparing intravesical mitomycin C versus bacillus Calmette Guérin for non-muscleinvasive bladder cancer. Eur Urol 29; 56: Whiteside TL: Inhibiting the inhibitors: evaluating agents targeting cancer immunosuppression. Expert Opin Biol Ther 21; 1: Gabrilovich DI, Nagaraj S: Myeloid-derived suppressor cells as regulators of the immune system. Nat Rev Immunol 29; 9: Diaz-Montero CM, Salem ML, Nishimura MI, et al: Increased circulating myeloid-derived suppressor cells correlate with clinical cancer stage, metastatic tumor burden, and doxorubicin cyclophosphamide chemo therapy. Cancer Immunol Immunother 29; 58: Li H, Han Y, Guo Q, et al: Cancer-expanded myeloid-derived suppressor cells induce anergy of NK cells through membrane-bound TGF-β1. J Immunol 29; 182: Pan PY, Ma G, Weber KJ, et al: Immune stimulatory receptor CD4 is required for T-cell suppression and T regulatory cell activation mediated by myeloid-derived suppressor cells in cancer. Cancer Res 21; 7: Serafini P, Mgebroff S, Noonan K, et al: Myeloidderived suppressor cells promote cross-tolerance in B-cell lymphoma by expanding regulatory T cells. Cancer Res 28; 68: Youn JI, Nagaraj S, Collazo M, et al: Subsets of myeloid-derived suppressor cells in tumorbearing mice. J Immunol 28; 181: Movahedi K, Guilliams M, Van den Bossche J, et al: Identification of discrete tumor-induced myeloid-derived suppressor cell subpopulations with distinct T cell-suppressive activity. Blood 28; 111: Ochoa AC, Zea AH, Hernandez C, et al: Arginase, prostaglandins, and myeloid-derived suppressor cells in renal cell carcinoma. Clin Cancer Res 27; 13: 721s 726s. 13 Almand B, Clark JI, Nikitina E, et al: Increased production of immature myeloid cells in cancer patients: a mechanism of immunosuppression in cancer. J Immunol 21; 166: Schmielau J, Finn OJ: Activated granulocytes and granulocyte-derived hydrogen peroxide are the underlying mechanism of suppression of T-cell function in advanced cancer patients. Cancer Res 21; 61: Zea AH, Rodriguez PC, Atkins MB, et al: Arginase-producing myeloid suppressor cells in renal cell carcinoma patients: a mechanism of tumor evasion. Cancer Res 25; 65: Filipazzi P, Valenti R, Huber V, et al: Identification of a new subset of myeloid suppressor cells in peripheral blood of melanoma patients with modulation by a granulocyte-macrophage colony-stimulation factor-based antitumor vaccine. J Clin Oncol 27; 25: Poschke I, Mougiakakos D, Hansson J, et al: Immature immunosuppressive CD14 + HLA- DR /low cells in melanoma patients are Stat3 hi and overexpress CD8, CD83, and DC-sign. Cancer Res 21; 7:

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